Methods and Compositions for Determining Altered Susceptibility of HIV-1 to Anti-HIV Drugs

ABSTRACT

This invention relates, in part, to methods and compositions for determining altered susceptibility of a human immunodeficiency virus (“HIV”) to the non-nucleoside reverse transcriptase inhibitors (“NNRTIs”) efavirenz (“EFV”), nevirapine (“NVP”), and delavirdine (“DLV”), the nucleoside reverse transcriptase inhibitor AZT, and the integrase strand transfer inhibitors diketo acid 1, diketo acid 2, and L-870,810 by detecting the presence of a mutation or combinations of mutations in the gene encoding HIV reverse transcriptase that are associated with altered susceptibility to the anti-HIV drugs.

PRIOR RELATED APPLICATIONS

This application is a divisional application of co-pending U.S.application Ser. No. 11/916,632 filed Dec. 5, 2007, which is a §371national phase application of PCT Application No. PCT/US06/22072 filedJun. 6, 2006, which claims priority to U.S. provisional application No.60/688,171 filed Jun. 6, 2005. Each of these applications is herebyincorporated by reference in its entirety.

1. FIELD OF INVENTION

This invention relates, in part, to methods and compositions fordetermining altered susceptibility of a human immunodeficiency virus(“HIV”) to the non-nucleoside reverse transcriptase inhibitors(“NNRTIs”) efavirenz (“EFV”), nevirapine (“NVP”), and delavirdine(“DLV”), the nucleoside reverse transcriptase inhibitor AZT, and theintegrase strand transfer inhibitors diketo acid 1, diketo acid 2, andL-870,810 by detecting the presence of a mutation or combinations ofmutations in an HIV pol gene that are associated with alteredsusceptibility to the anti-HIV drugs.

2. BACKGROUND OF THE INVENTION

More than 60 million people have been infected with the humanimmunodeficiency virus (“HIV”), the causative agent of acquired immunedeficiency syndrome (“AIDS”), since the early 1980s. See Lucas, 2002,Lepr Rev. 73(1):64-71. HIV/AIDS is now the leading cause of death insub-Saharan Africa, and is the fourth biggest killer worldwide. At theend of 2001, an estimated 40 million people were living with HIVglobally. See Norris, 2002, Radiol Technol. 73(4):339-363.

Modern anti-HIV drugs target different stages of the HIV life cycle anda variety of enzymes essential for HIV's replication and/or survival.Amongst the drugs that have so far been approved for AIDS therapy arenucleoside reverse transcriptase inhibitors (“NRTIs”) such as AZT, ddl,ddC, d4T, 3TC, and abacavir; nucleotide reverse transcriptase inhibitorssuch as tenofovir; non-nucleoside reverse transcriptase inhibitors(“NNRTIs”) such as nevirapine, efavirenz, and delavirdine; proteaseinhibitors (“PIs”) such as saquinavir, ritonavir, indinavir, nelfinavir,amprenavir, lopinavir and atazanavir; and fusion inhibitors, such asenfuvirtide. In addition, a number of drugs in other classes arecurrently under investigation for their ability to effectively treat HIVinfection. Among such drugs are integrase strand transfer inhibitors(“INSTIs”) such as the diketo acids diketo acid 1 and diketo acid 2 andthe napthyridine carboximides L-870,810 and MK0518.

Nonetheless, in the vast majority of subjects none of these antiviraldrugs, either alone or in combination, proves effective either toprevent eventual progression of chronic HIV infection to AIDS or totreat acute AIDS. This phenomenon is due, in part, to the high mutationrate of HIV and the rapid emergence of mutant HIV strains that areresistant to antiviral therapeutics upon administration of such drugs toinfected individuals.

Many such mutant strains have been characterized in order to correlatepresence of the mutations in the strains with resistant or susceptiblephenotypes. For example, the K103N mutation in reverse transcriptase isknown to correlate with resistance to a number of NNRTIs. See, e.g., DeClercq, 1997, Intl J. of Antimicrobial Agents 9:21-36. In addition, theP225H mutation in reverse transcriptase is also known to correlate withresistance to HIV-1 specific reverse transcriptase inhibitors (RTI).See, e.g., Pelemans et al., 1998, J. Gen. Virol. 79(Pt6):1347-52. Thus,a given mutation may correlate with resistance to one or more antiviralagents.

Though numerous mutations associated with resistance to particularanti-viral agents have been identified, the complete set of mutationsassociated with resistance to NNRTIs, to NRTIs, and to INSTIs has notbeen identified. Further, in view of the clinical relevance of NRTI,NNRTI, and INSTI resistance, a more complete understanding of mutationsassociated with such resistance is also needed. Thus, there remains aneed to identify additional mutations associated with resistance toNRTIs, NNRTIs, and INSTIs and to characterize these mutations. For thefirst time, these, as well as other unmet needs, will be achievable as aresult of the invention described hereinafter.

3. SUMMARY OF THE INVENTION

In certain aspects, the present invention provides methods fordetermining whether an HIV-1 is resistant to anti-HIV drugs, includingan NRTI, an NNRTI, or an INSTI. In the methods, resistance to ananti-HIV drug can be determined by detecting the presence of mutationsthat correlate with resistance to an anti-HIV drug.

Thus, in certain aspects, the invention provides a method fordetermining whether an HIV-1 is resistant to an NNRTI or to AZT,comprising detecting whether a mutation at codon 348 or 369 is presentin a gene encoding reverse transcriptase of the HIV-1, wherein thepresence of the mutation correlates with resistance to an NNRTI or toAZT, such that if the mutation at codon 348 or 369 is present, the HIV-1is resistant to the NNRTI. In certain embodiments, the methods comprisedetecting whether a mutation at codon 348 or 369 is present in the geneencoding reverse transcriptase in combination with a mutation at codon103, 179, 190, or 225, wherein the presence of the mutations correlateswith resistance to an NRTI, such that if the mutations are present, theHIV-1 is resistant to the NNRTI. In some embodiments, the methodscomprise detecting whether a mutation at codon 348 or 369 is present incombination with a mutation at codon 103. In other embodiments, themethods comprise detecting whether a mutation at codon 369 is present incombination with a mutation at codon 225. In some embodiments, themethods comprise detecting whether a mutation at codon 348 or 369 ispresent in combination with a mutation at codon 190. In someembodiments, the methods comprise detecting whether a mutation at codon348 or 369 is present in combination with a mutation at codon 103 and atcodon 179. In another embodiment, the methods comprise detecting whethera mutation at codon 369 is present in combination with a mutation atcodon 103 and a mutation at codon 225, wherein the presence of themutations correlates with resistance to an NNRTI, such that if themutations are present, the HIV-1 is resistant to the NNRTI.

In yet other embodiments, the method comprises detecting whether amutation at codon 399 in combination with a mutation at codon 103, 179,or 190 is present in a gene encoding reverse transcriptase of the HIV-1,wherein the presence of the mutations correlates with resistance to anNNRTI, such that if the mutation is present, the HIV-1 is resistant tothe NNRTI. In some embodiments, the methods comprise detecting whether amutation at codon 399 is present in combination with a mutation at codon190. In some embodiments, In some embodiments, the methods comprisedetecting whether a mutation at codon 399 is present in combination witha mutation at codon 103. In some embodiments, In some embodiments, themethods comprise detecting whether a mutation at codon 399 is present incombination with a mutation at codon 103 and at codon 179.

In another aspect, the invention provides a method for determiningwhether an HIV-1 has reduced replication capacity relative to areference HIV-1, comprising detecting, in a nucleic acid encodingreverse transcriptase of the HIV-1, a mutation at codon 369 or in anucleic acid encoding integrase of the HIV-1, a mutation at codon 97,wherein the presence of the mutation correlates with reduced replicationcapacity such that if the mutation is present, the HIV-1 has reducedreplication capacity relative to a reference HIV-1. In certainembodiments, a mutation at codon 369 of reverse transcriptase isdetected. In certain embodiments, a mutation at codon 97 of integrase isdetected.

In still another aspect, the invention provides a method for determiningwhether a human immunodeficiency virus 1 (HIV-1) has alteredsusceptibility to a integrase strand transfer inhibitor (INSTI),comprising detecting whether a mutation at codon 97 or codon 156 ispresent in a gene encoding integrase of the HIV-1, wherein the presenceof the mutations correlates with altered susceptibility to an INSTI,such that if the mutation is present, the HIV-1 is resistant to theINSTI. In certain embodiments, a mutation at codon 97 is detected. Incertain embodiments, a mutation at codon 156 is detected. In certainembodiments, the HIV-1 exhibits increased susceptibility to the INSTI.In certain embodiments, the HIV-1 exhibits decreased susceptibility tothe INSTI.

In certain embodiments, the method further comprises detecting whether amutation at codon 66, 72, 121, 125, 154, or 155 is present in the geneencoding integrase. In certain embodiments, a mutation at codon 66 isdetected. In certain embodiments, a mutation at codon 72 is detected. Incertain embodiments, a mutation at codon 121 is detected. In certainembodiments, a mutation at codon 125 is detected. In certainembodiments, a mutation at codon 154 is detected. In certainembodiments, a mutation at codon 155 is detected. In certainembodiments, mutations at codons 72, 121, and 125 are detected. Incertain embodiments, mutations at codons 66 and 154 are detected.

The presence of the mutations associated with resistance to AZT, to anNNRTI, or to an INSTI can be detected according to any method known toone of skill in the art without limitation. Methods for detecting suchmutations are described extensively below.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates that there are no statistical differences in NNRTIIC₅₀ fold change values obtained in phenotypic assays using resistancetest vectors comprising POL and RHIN sequences. Resistance assays wereperformed using different resistance test vectors comprising differentpatient-derived segments (POL and PR-RT) from the same HIV-infectedpatient samples to assess relative contributions of the segments toresistance to NNRTIs (NVP, DLV and EFV) of HIV isolated fromHIV-infected patients. Results (fold change in IC50 or “FC”) for each ofthese different amplicons prepared from the same patient samples werecompared.

FIG. 2 provides a diagrammatic representation of constructs fordifferent amplicons of sample 62 tested in FIG. 3. The POL ampliconcomprises a patient-derived segment corresponding to the entire poigene, encoding HIV protease, reverse transcriptase (including RNase H),and integrase. The PR-RT amplicon comprises a patient-derived segmentcorresponding to the portion of pol encoding HIV protease and aminoacids 1-305 of reverse transcriptase. The RHIN amplicon comprises apatient-derived segment corresponding to the portion of pol encodingamino acids 319-440 of reverse transcriptase (including RNase H), andintegrase. The PRRT-RH amplicon comprises a patient-derived segmentcorresponding to the portion of pol encoding protease and amino acids1-531 of reverse transcriptase (including RNase H). The PRRT-RHINamplicon comprises a patient-derived segment corresponding to theportion of pol encoding protease, reverse transcriptase (including RNaseH) and integrase.

FIG. 3 shows that different amplicons and constructs derived from apatient sample exhibit different degrees of NVP resistance.

FIG. 4 presents the fold change in IC50 observed in the presence of NVPfor an HIV-1 site-directed mutant that comprises the T369I mutation inreverse transcriptase in a NL4-3 background.

FIG. 5 presents the fold change in IC50 observed in the presence of NVPfor an HIV-1 site-directed mutant that comprises the T369I mutation andthe K103N mutation in reverse transcriptase in a NL4-3 background.

FIG. 6 presents the fold change in IC50 observed in the presence of NVPfor the PRRT patient-derived segment obtained from sample 62 and alsocomprising T369I site-directed mutation.

FIG. 7 presents comparisons between the fold change in NNRTI resistanceobserved for the PR-RT patient segment and the POL patient segment for52 patient samples.

FIG. 8 presents the distribution of fold change in NNRTI resistance forpatient samples having mutations at RT positions 348, 369, and 399.

FIG. 9 presents the results of phenotypic assays showing resistance orsusceptibility to NNRTIs on site-directed mutants having mutations at RTpositions 348, 369, and 399 in an NL4-3 background.

FIG. 10 presents the results of phenotypic assays showing resistance orsusceptibility to NNRTIs on site-directed mutants having mutations at RTpositions 348, 369, and 399 in combination with previously identifiedNNRTI resistance mutations in an NL4-3 background.

FIG. 11 presents the results of phenotypic assays showing resistance orsusceptibility to NNRTIs on site-directed mutants having mutations at RTpositions 348, 369, and 399 in three different patient backgrounds.

FIG. 12 presents comparisons between the fold change in AZT resistanceobserved for the PR-RT patient segment and the POL patient segment for52 patient samples.

FIG. 13 presents the results of phenotypic assays showing resistance orsusceptibility to AZT on site-directed mutants having mutations at RTpositions 348, 369, and 399 in an NL4-3 background.

FIG. 14 presents the results of phenotypic assays showing resistance orsusceptibility to AZT on site-directed mutants having mutations at RTpositions 348, 369, and 399 in two different patient backgrounds.

FIG. 15 presents the results of phenotypic assays showing that the RTmutation T369I causes a reduction in replication capacity in an NL4-3background.

FIG. 16 presents the results of phenotypic assays showing that RTmutations in codons 348, 369, and 399 cause a reduction in replicationcapacity in some patient backgrounds.

FIG. 17 presents the distribution of resistance or susceptibility inIC50 fold change to the INSTI L-870,810 in 128 patient samples.

FIG. 18 presents the results of phenotypic assays showing that mutationsin integrase codons 97 and 156 result in altered susceptibility to theINSTI L-870,810, but do not result in altered susceptibility to theINSTIs diketo acid 1 and diketo acid 2.

FIG. 19 presents the results of phenotypic assays showing that INmutations in codon 97 cause a reduction in replication capacity in anNL4-3 background.

FIG. 20 presents a table containing the results of phenotypic assaysshowing that mutations in integrase codons 97 and 156 in combinationwith previously recognized INSTI resistance mutations result in alteredsusceptibility to the INSTIs L-diketo acid 1, diketo acid 2, andL-870,810 and in reduced replication capacity.

5. DETAILED DESCRIPTION OF THE INVENTION

In certain aspects, the present invention provides methods fordetermining whether an HIV-1 is resistant to antiviral therapy with anNNRTI or with AZT. The methods generally comprise detecting whether amutation or mutations are present in the HIV-1 gene encoding RT thatsignificantly correlate with resistance to an NNRTI or to AZT.

In other aspects, the present invention provides methods for determiningwhether an HIV-1 has reduced replication capacity. The methods generallycomprise detecting whether a mutation or mutations are present in theHIV-1 gene encoding RT that significantly correlate with reducedreplication capacity.

In still other aspects, the present invention provides methods fordetermining whether an HIV-1 has altered susceptibility to antiviraltherapy with an INSTI. The methods generally comprise detecting whethera mutation or mutations are present in the HIV-1 gene encoding IN thatsignificantly correlate with altered susceptibility to an INSTI.

5.1 Abbreviations

“NRTI” is an abbreviation for nucleoside reverse transcriptaseinhibitor.

“NNRTI” is an abbreviation for non nucleoside reverse transcriptaseinhibitor.

“PI” is an abbreviation for protease inhibitor.

“PR” is an abbreviation for protease.

“RT” is an abbreviation for reverse transcriptase.

“IN” is an abbreviation for integrase.

“PCR” is an abbreviation for “polymerase chain reaction.”

“HBV” is an abbreviation for hepatitis B virus.

“HCV” is an abbreviation for hepatitis C virus.

“HIV” is an abbreviation for human immunodeficiency virus.

“EFV” is an abbreviation for the NNRTI efavirenz.

“DLV” is an abbreviation for the NNRTI delavirdine.

“NVP” is an abbreviation for the NNRTI nevirapine.

“INSTI” is an abbreviation for a integrase strand transfer inhibitor.

The amino acid notations used herein for the twenty genetically encodedL-amino acids are conventional and are as follows:

One-Letter Three Letter Amino Acid Abbreviation Abbreviation Alanine AAla Arginine R Arg Asparagine N Asn Aspartic acid D Asp Cysteine C CysGlutamine Q Gln Glutamic acid E Glu Glycine G Gly Histidine H HisIsoleucine I Ile Leucine L Leu Lysine K Lys Methionine M MetPhenylalanine F Phe Proline P Pro Serine S Ser Threonine T ThrTryptophan W Trp Tyrosine Y Tyr Valine V Val

Unless noted otherwise, when polypeptide sequences are presented as aseries of one-letter and/or three-letter abbreviations, the sequencesare presented in the N→C direction, in accordance with common practice.

Individual amino acids in a sequence are represented herein as AN,wherein A is the standard one letter symbol for the amino acid in thesequence, and N is the position in the sequence. Mutations arerepresented herein as A1NA2, wherein A₁ is the standard one lettersymbol for the amino acid in the reference protein sequence, A2 is thestandard one letter symbol for the amino acid in the mutated proteinsequence, and N is the position in the amino acid sequence. For example,a G25M mutation represents a change from glycine to methionine at aminoacid position 25. Mutations may also be represented herein as NA2,wherein N is the position in the amino acid sequence and A2 is thestandard one letter symbol for the amino acid in the mutated proteinsequence (e.g., 25M, for a change from the wild-type amino acid tomethionine at amino acid position 25). Additionally, mutations may alsobe represented herein as A1NX, wherein A1 is the standard one lettersymbol for the amino acid in the reference protein sequence, N is theposition in the amino acid sequence, and X indicates that the mutatedamino acid can be any amino acid (e.g., G25X represents a change fromglycine to any amino acid at amino acid position 25). This notation istypically used when the amino acid in the mutated protein sequence iseither not known or, if the amino acid in the mutated protein sequencecould be any amino acid, except that found in the reference proteinsequence. The amino acid positions are numbered based on the full-lengthsequence of the protein from which the region encompassing the mutationis derived. Representations of nucleotides and point mutations in DNAsequences are analogous.

The abbreviations used throughout the specification to refer to nucleicacids comprising specific nucleobase sequences are the conventionalone-letter abbreviations. Thus, when included in a nucleic acid, thenaturally occurring encoding nucleobases are abbreviated as follows:adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U).Unless specified otherwise, single-stranded nucleic acid sequences thatare represented as a series of one-letter abbreviations, and the topstrand of double-stranded sequences, are presented in the 5′→3′direction.

5.2 Definitions

As used herein, the following terms shall have the following meanings:

A “phenotypic assay” is a test that measures a phenotype of a particularvirus, such as, for example, HIV, or a population of viruses, such as,for example, the population of HIV infecting a subject. The phenotypesthat can be measured include, but are not limited to, the resistance orsusceptibility of a virus, or of a population of viruses, to a specificanti-viral agent or that measures the replication capacity of a virus.

A “genotypic assay” is an assay that determines a genotype of anorganism, a part of an organism, a population of organisms, a gene, apart of a gene, or a population of genes. Typically, a genotypic assayinvolves determination of the nucleic acid sequence of the relevant geneor genes. Such assays are frequently performed in HIV to establish, forexample, whether certain mutations are associated with drug resistanceor hypersusceptibility or altered replication capacity are present.

As used herein, “genotypic data” are data about the genotype of, forexample, a virus. Examples of genotypic data include, but are notlimited to, the nucleotide or amino acid sequence of a virus, apopulation of viruses, a part of a virus, a viral gene, a part of aviral gene, or the identity of one or more nucleotides or amino acidresidues in a viral nucleic acid or protein.

The term “% sequence identity” is used interchangeably herein with theterm “% identity” and refers to the level of amino acid sequenceidentity between two or more peptide sequences or the level ofnucleotide sequence identity between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% identity means the same thing as 80% sequence identitydetermined by a defined algorithm, and means that a given sequence is atleast 80% identical to another length of another sequence. Exemplarylevels of sequence identity include, but are not limited to, 60, 70, 80,85, 90, 95, 98% or more sequence identity to a given sequence.

The term “% sequence homology” is used interchangeably herein with theterm “% homology” and refers to the level of amino acid sequencehomology between two or more peptide sequences or the level ofnucleotide sequence homology between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% homology means the same thing as 80% sequence homologydetermined by a defined algorithm, and accordingly a homologue of agiven sequence has greater than 80% sequence homology over a length ofthe given sequence. Exemplary levels of sequence homology include, butare not limited to, 60, 70, 80, 85, 90, 95, 98% or more sequencehomology to a given sequence.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet at the NCBI website. See alsoAltschul et al., 1990, J. Mol. Biol. 215:403-10 (with special referenceto the published default setting, i.e., parameters w=4, t=17) andAltschul et al., 1997, Nucleic Acids Res., 25:3389-3402. Sequencesearches are typically carried out using the BLASTP program whenevaluating a given amino acid sequence relative to amino acid sequencesin the GenBank Protein Sequences and other public databases. The BLASTXprogram is preferred for searching nucleic acid sequences that have beentranslated in all reading frames against amino acid sequences in theGenBank Protein Sequences and other public databases. Both BLASTP andBLASTX are run using default parameters of an open gap penalty of 11.0,and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix.See id.

A preferred alignment of selected sequences in order to determine “%identity” between two or more sequences, is performed using for example,the CLUSTAL-X program, operated with default parameters, including anopen gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM30 similarity matrix.

“Polar Amino Acid” refers to a hydrophilic amino acid having a sidechain that is uncharged at physiological pH, but which has at least onebond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Genetically encoded polar aminoacids include Asn (N), Gln (Q) Ser (S) and Thr (T).

“Nonpolar Amino Acid” refers to a hydrophobic amino acid having a sidechain that is uncharged at physiological pH and which has bonds in whichthe pair of electrons shared in common by two atoms is generally heldequally by each of the two atoms (i.e., the side chain is not polar).Genetically encoded nonpolar amino acids include Ala (A), Gly (G), Ile(I), Leu (L), Met (M) and Val (V).

“Hydrophilic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of less than zero according to the normalized consensushydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophilic amino acids include Arg(R), Asn (N), Asp (D), Glu (E), Gln (Q), His (H), Lys (K), Ser (S) andThr (T).

“Hydrophobic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophobic amino acids include Ala(A), Gly (G), Ile (I), Leu (L), Met (M), Phe (F), Pro (P), Trp (W), Tyr(Y) and Val (V).

“Acidic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Asp (D) andGlu (E).

“Basic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith a hydrogen ion. Genetically encoded basic amino acids include Arg(R), His (H) and Lys (K).

A “mutation” is a change in an amino acid sequence or in a correspondingnucleic acid sequence relative to a reference nucleic acid orpolypeptide. For embodiments of the invention comprising HIV protease orreverse transcriptase, the reference nucleic acid encoding protease orreverse transcriptase is the protease or reverse transcriptase codingsequence, respectively, present in NL4-3 HIV (SEQ ID NO:5; GenBankAccession No. AF324493). Likewise, the reference protease or reversetranscriptase polypeptide is that encoded by the NL4-3 HIV sequence.Although the amino acid sequence of a peptide can be determined directlyby, for example, Edman degradation or mass spectroscopy, more typically,the amino sequence of a peptide is inferred from the nucleotide sequenceof a nucleic acid that encodes the peptide. Any method for determiningthe sequence of a nucleic acid known in the art can be used, forexample, Maxam-Gilbert sequencing (Maxam et al., 1980, Methods inEnzymology 65:499), dideoxy sequencing (Sanger et al., 1977, Proc. Natl.Acad. Sci. USA 74:5463) or hybridization-based approaches (see e.g.,Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, 3.sup.rd ed., NY; and Ausubel et al., 1989,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, NY).

A “mutant” is a virus, gene or protein having a sequence that has one ormore changes relative to a reference virus, gene or protein.

The terms “peptide,” “polypeptide” and “protein” are usedinterchangeably throughout.

The term “wild-type” refers to a viral genotype that does not comprise amutation known to be associated with drug resistance.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout.

5.3 Methods of Determining Altered Susceptibility to an NRTI, NNRTI, orINSTI

In certain aspects, the present invention provides methods fordetermining whether an HIV-1 is has altered susceptibility to an NRTI,NNRTI, or INSTI. In general, the methods comprise detecting whethermutations significantly correlated with altered susceptibility to anNRTI, NNRTI, or INSTI are present in the gene encoding reversetranscriptase or integrase of the HIV-1, as demonstrated by the examplesbelow. In certain embodiments, the HIV-1 has increased susceptibility,e.g., hypersusceptibility, to the NRTI, NNRTI, or INSTI. In certainembodiments, the HIV-1 has decreased susceptibility, e.g., is resistant,to the NRTI, NNRTI, or INSTI.

In certain embodiments, viruses that exhibit an IC50 2.5 fold higherthan wild-type virus were designated as having resistance to an NRTI,NNRTI, or INSTI. In some embodiments, viruses that exhibit an IC₅0 2.0fold higher than wild-type virus were designated as having resistance toan NRTI, NNRTI, or INSTI. In some embodiments, viruses that exhibit anIC₅0 1.5 fold higher than wild-type virus were designated as havingresistance to an NRTI, NNRTI, or INSTI. In certain embodiments, virusesthat exhibit an IC50 2.5 fold lower than wild-type virus were designatedas having hypersusceptibility to an NRTI, NNRTI, or INSTI. In someembodiments, viruses that exhibit an IC50 2.0 fold lower than wild-typevirus were designated as having hypersusceptibility to an NRTI, NNRTI,or INSTI. In some embodiments, viruses that exhibit an IC50 1.5 foldlower than wild-type virus were designated as having hypersusceptibilityto an NRTI, NNRTI, or INSTI.

Therefore, in certain embodiments, the invention provides a method fordetermining whether a human immunodeficiency virus 1 (HIV-1) isresistant to a non-nucleoside reverse transcriptase inhibitor (NNRTI) orziduvine (AZT), comprising detecting whether a mutation at codon 348 or369 is present in a gene encoding reverse transcriptase of the HIV-1,wherein the presence of the mutation correlates with resistance to anNNRTI or to AZT, such that if the mutation is present, the HIV-1 isresistant to the NNRTI or to AZT. In certain embodiments, the mutationat codon 348 encodes isoleucine (I). In certain embodiments, themutation at codon 369 encodes isoleucine (I) or alanine (A). In certainembodiments, the NNRTI is efavirenz (EFV), nevirapine (NVP), ordelavirdine (DLV). In certain embodiments, the NNRTI is EFV. In certainembodiments, the NNRTI is NVP. In certain embodiments, the NNRTI is DLV.In certain embodiments, the HIV-1 is determined to be resistant to AZT.

In certain embodiments, the methods further comprise detecting whether amutation at codon 103, 179, 190, or 225 is present in the gene encodingreverse transcriptase. In certain embodiments, a mutation at codon 103is detected. In certain embodiments, the mutation at codon 103 encodesasparagine (N), arginine (R), serine (S), glutamine (Q), or threonine(T). In certain embodiments, a mutation at codon 225 is detected. Incertain embodiments, the mutation at codon 225 encodes histidine (H). Incertain embodiments, a mutation at codon 103 and a mutation at codon 225are detected. In certain embodiments, the mutation at codon 103 encodesasparagine (N), arginine (R), serine (S), glutamine (Q), or threonine(T). In certain embodiments, the mutation at codon 225 encodes histidine(H). In certain embodiments, a mutation at codon 190 is detected. Incertain embodiments, the mutation at codon 190 encodes serine (S). Incertain embodiments, mutations at codon 103 and codon 179 are detected.In certain embodiments, the mutation at codon 103 encodes asparagine(N), arginine (R), serine (S), glutamine (Q), or threonine (T). Incertain embodiments, the mutation at codon 179 encodes aspartic acid(D). In certain embodiments, the NNRTI is EFV, NVP, or DLV. In certainembodiments, the NNRTI is EFV. In certain embodiments, the NNRTI is NVP.In certain embodiments, the NNRTI is DLV.

In another aspect, the invention provides a method for determiningwhether a human immunodeficiency virus 1 (HIV-1) is resistant to anon-nucleoside reverse transcriptase. inhibitor (NNRTI), comprisingdetecting whether a mutation at codon 399 in combination with a mutationat codon 103, 179, or 190 is present in a gene encoding reversetranscriptase of the HIV-1, wherein the presence of the mutationscorrelates with resistance to an NNRTI, such that if the mutation ispresent, the HIV-1 is resistant to the NNRTI. In certain embodiments,the mutation at codon 399 encodes aspartic acid (D). In certainembodiments, a mutation at codon 103 is detected. In certainembodiments, the mutation at codon 103 encodes asparagine (N), arginine(R), serine (S), glutamine (Q), or threonine (T). In certainembodiments, a mutation at codon 179 is detected. In certainembodiments, the mutation at codon 179 encodes aspartic acid (D). Incertain embodiments, a mutation at codon 190 is detected. In certainembodiments, the mutation at codon 190 encodes serine (S). In certainembodiments, mutations at codons 103 and 179 are detected. In certainembodiments, the mutation at codon 103 encodes asparagine (N), arginine(R), serine (S), glutamine (Q), or threonine (T). In certainembodiments, the mutation at codon 179 encodes aspartic acid (D). Incertain embodiments, the NNRTI is EFV, NVP, or DLV. In certainembodiments, the NNRTI is EFV. In certain embodiments, the NNRTI is NVP.In certain embodiments, the NNRTI is DLV.

In another aspect, the invention provides a method for determiningwhether an HIV-1 has reduced replication capacity relative to areference HIV-1, comprising detecting, in a nucleic acid encodingreverse transcriptase of the HIV-1, a mutation at codon 369 or in anucleic acid encoding integrase of the HIV-1, a mutation at codon 97,wherein the presence of the mutation correlates with reduced replicationcapacity such that if the mutation is present, the HIV-1 has reducedreplication capacity relative to a reference HIV-1. In certainembodiments, a mutation at codon 369 of reverse transcriptase isdetected. In certain embodiments, the mutation at codon 369 encodesisoleucine (I). In certain embodiments, a mutation at codon 97 ofIntegrase is detected. In certain embodiments, the mutation at codon 97encodes alanine (A). In certain embodiments, the reference HIV-1 isNL4-3. In certain embodiments, the reference HIV-1 has an identicalgenotype to the HIV-1 having its replication capacity determined exceptfor codon 369 of reverse transcriptase or codon 97 of integrase. Incertain embodiments, the genotype of the reference HIV-1 differs fromthe HIV-1 having its replication capacity determined at codon 369 ofreverse transcriptase. In certain embodiments, the genotype of thereference HIV-1 differs from the HIV-1 having its replication capacitydetermined at codon 97 of integrase.

In yet another aspect, the invention provides a method for determiningwhether a human immunodeficiency virus 1 (HIV-1) has alteredsusceptibility to a integrase strand transfer inhibitor (INSTI),comprising detecting whether a mutation at codon 97 or codon 156 ispresent in a gene encoding integrase of the HIV-1, wherein the presenceof the mutations correlates with altered susceptibility to an INSTI,such that if the mutation is present, the HIV-1 has alteredsusceptibility to the INSTI. In certain embodiments, the HIV-1 exhibitsincreased susceptibility to the INSTI. In certain embodiments, the HIV-1exhibits decreased susceptibility to the INSTI. In certain embodiments,a mutation at codon 97 is detected. In certain embodiments, the mutationat codon 97 encodes alanine (A). In certain embodiments, a mutation atcodon 156 is detected. In certain embodiments, the mutation at codon 156encodes asparagines (N). In certain embodiments, the INSTI is anapthyridine carboximide. In certain embodiments, the INSTI isL-870,810.

In certain embodiments, the methods further comprise detecting whether amutation at codon 66, 72, 121, 125, 154, or 155 is present in the geneencoding integrase. In certain embodiments, the HIV-1 exhibits decreasedsusceptibility to the INSTI. In certain embodiments, a mutation at codon66 is detected. In certain embodiments, the mutation at codon 66 encodesisoleucine (I). In certain embodiments, a mutation at codon 72 isdetected. In certain embodiments, the mutation at codon 72 encodesisoleucine (I). In certain embodiments, a mutation at codon 121 isdetected. In certain embodiments, the mutation at codon 121 encodestyrosine (Y). In certain embodiments, a mutation at codon 125 isdetected. In certain embodiments, the mutation at codon 125 encodeslysine (K). In certain embodiments, a mutation at codon 154 is detected.In certain embodiments, the mutation at codon 154 encodes isoleucine(I). In certain embodiments, a mutation at codon 155 is detected. Incertain embodiments, the mutation at codon 155 encodes serine (S). Incertain embodiments, mutations at codons 72, 121, and 125 are detected.In certain embodiments, the mutation at codon 72 encodes isoleucine (I),the mutation at codon 121 encodes tyrosine (Y), and the mutation atcodon 125 encodes lysine (K). In certain embodiments, mutations atcodons 66 and 154 are detected. In certain embodiments, the mutation atcodon 66 encodes isoleucine (I) and the mutation at codon 154 encodesisoleucine (I). In certain embodiments, the INSTI is a diketo acid. Incertain embodiments, the INSTI is diketo acid 1 or diketo acid 2. Incertain embodiments, the INSTI is a napthyridine carboximide. In certainembodiments, the INSTI is L-870,810.

In another aspect, the methods comprise determining whether a subject isinfected with an HIV that is sensitive to an NRTI, NNRTI, or INSTIaccording to a method of the invention, then advising a medicalprofessional of the treatment option of administering to the subject aneffective amount of an NRTI, NNRTI, or INSTI, respectively. In anotheraspect, the methods comprise determining whether a subject is infectedwith an HIV that is resistant to an NRTI, NNRTI, or INSTI according to amethod of the invention, then advising a medical professional of thetreatment option of administering to the subject an effective amount ofan NRTI, NNRTI, or INSTI, respectively. Preferably, the HIV that isresistant to the NRTI, NNRTI, or INSTI comprises a mutation associatedwith NRTI, NNRTI, or INSTI resistance, respectively, that is associatedwith impaired replication. Such mutations are described herein and inU.S. Pub. No. 2004/0063191 and U.S. application Ser. Nos. 11/052,741 and11/092,204, which are incorporated by reference in their entireties. Incertain embodiments, the NRTI is AZT. In certain embodiments, the NNRTIis EFV, DLV, or NVP. In certain embodiments, the INSTI is diketo acid 1,diketo acid 2 or L-870,810. In some embodiments, the methods comprisedetermining whether a subject is infected with an HIV that is resistantto an NRTI, NNRTI, or INSTI according to a method of the invention, thenadvising a medical professional of the option not to treat the subjectwith an NRTI, NNRTI, or INSTI, respectively. In certain embodiments, theNRTI is AZT. In certain embodiments, the NNRTI is EFV, DLV, or NVP. Incertain embodiments, the INSTI is diketo acid 1, diketo acid 2 orL-870,810.

In still another aspect, the methods comprise determining that a subjectis infected with an HIV that is resistant to an NRTI, NNRTI, or INSTIaccording to a method of the invention, and administering to the subjecta combination of anti-HIV agents that comprises an effective amount ofan NRTI, NNRTI, or INSTI, respectively. Preferably, the HIV that isresistant to the NRTI, NNRTI, or INSTI comprises a mutation associatedwith NRTI, NNRTI, or INSTI resistance, respectively, that is associatedwith impaired replication, as discussed above. In certain embodiments,the NRTI is AZT. In certain embodiments, the NNRTI is EFV, DLV, or NVP.In certain embodiments, the INSTI is diketo acid 1, diketo acid 2 orL-870,810. In some embodiments, the methods comprise determining that asubject is infected with an HIV that is resistant to an NRTI, NNRTI, orINSTI according to a method of the invention, and administering to thesubject a combination of anti-HIV agents that does not comprise theNRTI, NNRTI, or INSTI, respectively. In certain embodiments, the NRTI isAZT. In certain embodiments, the NNRTI is EFV, DLV, or NVP. In certainembodiments, the INSTI is diketo acid 1, diketo acid 2 or L-870,810.

In still another aspect, the methods comprise determining that a subjectis infected with an HIV that is resistant to an NRTI, NNRTI, or INSTIaccording to a method of the invention at a first time, then determiningwhether the subject remains infected with an HIV that is resistant to anNRTI, NNRTI, or INSTI according to a method of the invention at a latersecond time. Preferably, the subject has undergone or has beenundergoing an anti-HIV therapy during the period between the first andsecond time. In other embodiments, the methods comprise determining thata subject is infected with an HIV that is sensitive to an NRTI, NNRTI,or INSTI according to a method of the invention at a first time, thendetermining whether the subject is infected with an HIV that isresistant to an NRTI, NNRTI, or INSTI according to a method of theinvention at a later second time. Preferably, the subject has undergoneor has been undergoing an anti-HIV therapy that comprises an effectiveamount of an NRTI, NNRTI, or INSTI during the period between the firstand second time.

5.4 Measuring Resistance of HIV-1 to an Anti-Viral Drug

Any method known in the art can be used to determine a viral drugresistance phenotype, without limitation. See e.g., U.S. Pat. Nos.5,837,464 and 6,242,187, each of which is hereby incorporated byreference in its entirety.

In certain embodiments, the phenotypic analysis is performed usingrecombinant virus assays (“RVAs”). RVAs use virus stocks generated byhomologous recombination between viral vectors and viral gene sequences,amplified from the patient virus. In certain embodiments, the viralvector is a HIV vector and the viral gene sequences are protease and/orreverse transcriptase and/or gag sequences.

In preferred embodiments, the phenotypic analysis of NRTI, NNRTI, orINSTI resistance is performed using PHENOSENSE® (Monogram Biosciences,Inc., South San Francisco, Calif.). See Petropoulos et al., 2000,Antimicrob. Agents Chemother. 44:920-928; U.S. Pat. Nos. 5,837,464 and6,242,187. PHENOSENSE® is a phenotypic assay that achieves the benefitsof phenotypic testing and overcomes the drawbacks of previous assays.Because the assay has been automated, PHENOSENSE® provides highthroughput methods under controlled conditions for determining NRTI,NNRTI, or INSTI resistance, susceptibility, or hypersusceptibility of alarge number of individual viral isolates.

The result is an assay that can quickly and accurately define both thereplication capacity and the susceptibility profile of a patient's HIV(or other virus) isolates to all currently available antiretroviraldrugs, including the NRTI AZT, the NNRTIs EFV, DLV, and NVP, and theINSTIs diketo acid 1, diketo acid 2, and L-870,810. PHENOSENSE® canobtain results with only one round of viral replication, therebyavoiding selection of subpopulations of virus that can occur duringpreparation of viral stocks required for assays that rely on fullyinfectious virus. Further, the results are both quantitative, measuringvarying degrees of replication capacity or antiviral resistance orsusceptibility, and sensitive, as the test can be performed on bloodspecimens with a viral load of about 500 copies/mL or above and candetect minority populations of some drug-resistant virus atconcentrations of 10% or less of total viral population. Finally, thereplication capacity and antiviral drug resistance results arereproducible and can vary by less than about 0.25 logs in about 95% ofthe assays performed.

PHENOSENSE® can be used with nucleic acids from amplified viral genesequences. As discussed below, the nucleic acid can be amplified fromany sample known by one of skill in the art to contain a viral genesequence, without limitation. For example, the sample can be a samplefrom a human or an animal infected with the virus or a sample from aculture of viral cells. In certain embodiments, the viral samplecomprises a genetically modified laboratory strain. In otherembodiments, the viral sample comprises a wild-type isolate.

A resistance test vector (“RTV”) can then be constructed byincorporating the amplified viral gene sequences into a replicationdefective viral vector by using any method known in the art ofincorporating gene sequences into a vector. In one embodiment,restrictions enzymes and conventional cloning methods are used. SeeSambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, 3^(rd) ed., NY; and Ausubel et al., 1989,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, NY. In a preferred embodiment, ApaI and PinAlrestriction enzymes are used. Preferably, the replication defectiveviral vector is the indicator gene viral vector (“IGVV”). In a preferredembodiment, the viral vector contains a means for detecting replicationof the RTV. Preferably, the viral vector contains a luciferaseexpression cassette.

The assay can be performed by first co-transfecting host cells with RTVDNA and a plasmid that expresses the envelope proteins of anotherretrovirus, for example, amphotropic murine leukemia virus (MLV).Following transfection, viral particles can be harvested from the cellculture and used to infect fresh target cells in the presence of varyingamounts of antiviral drug(s). The completion of a single round of viralreplication in the fresh target cells can be detected by the means fordetecting replication contained in the vector. In a preferredembodiment, the completion of a single round of viral replicationresults in the production of luciferase. By monitoring the amount of,e.g., luciferase activity in the presence of the varying amounts ofantiviral drug(s), a resistance curve can be constructed by plottingluciferase activity versus drug concentration. The resistance of an HIV,or population of HIV, can be determined by measuring the concentrationof antiviral drug at which the luciferase activity detected ishalf-maximal. This number, the IC₅₀, provides a standard and convenientmeasure of drug resistance.

In preferred embodiments, PHENOSENSE® is used to evaluate the AZT, EFV,DLV, NVP, diketo acid 1, diketo acid 2, and/or L-870,810 resistancephenotype of HIV-1. In other embodiments, PHENOSENSE® is used toevaluate the AZT, EFV, DLV, NVP, diketo acid 1, diketo acid 2, and/orL-870,810 resistance phenotype of HIV-2. In certain embodiments, theHIV-1 strain that is evaluated is a wild-type isolate of HIV-1. In otherembodiments, the HIV-1 strain that is evaluated is a mutant strain ofHIV-1. In certain embodiments, such mutant strains can be isolated frompatients. In other embodiments, the mutant strains can be constructed bysite-directed mutagenesis or other equivalent techniques known to one ofskill in the art. In still other embodiments, the mutant strains can beisolated from cell culture. The cultures can comprise multiple passagesthrough cell culture in the presence of antiviral compounds to selectfor mutations that accumulate in culture in the presence of suchcompounds.

In one embodiment, viral nucleic acid, for example, HIV-1 RNA isextracted from plasma samples, and a fragment of, or entire viral genescan be amplified by methods such as, but not limited to PCR. See, e.g.,Hertogs et aL, 1998, Antimicrob Agents Chemother 42(2):269-76. In oneexample, a 2.2-kb fragment containing the entire HIV-1 PR- and RT-codingsequence is amplified by nested reverse transcription-PCR. The pool ofamplified nucleic acid, for example, the PR-RT-coding sequences, is thencotransfected into a host cell such as CD4+ T lymphocytes (MT4) with thepGEMT3deltaPRT plasmid from which most of the PR (codons 10 to 99) andRT (codons 1 to 482) sequences are deleted. Homologous recombinationleads to the generation of chimeric viruses containing viral codingsequences, such as the PR- and RT-coding sequences derived from HIV-1RNA in plasma. Alternately, other patient segments can be amplified asdescribed, for example, in Example 2, below. The replication capacitiesor antiviral drug resistance phenotypes of the chimeric viruses can bedetermined by any cell viability assay known in the art, and compared toreplication capacities or antiviral drug resistance of a statisticallysignificant number of individual viral isolates to assess whether avirus has altered replication capacity or is resistant orhypersusceptible to the antiviral drug. For example, an MT4cell-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-basedcell viability assay can be used in an automated system that allows highsample throughput.

In another embodiment, competition assays can be used to assessreplication capacity of one viral strain relative to another viralstrain. For example, two infectious viral strains can be co-cultivatedtogether in the same culture medium. See, e.g., Lu et al., 2001, JAIDS27:7-13, which is incorporated by reference in its entirety. Bymonitoring the course of each viral strain's growth, the fitness of onestrain relative to the other can be determined. By measuring manyviruses' fitness relative to a single reference virus, an objectivemeasure of each strain's fitness can be determined.

Other assays for evaluating the phenotypic susceptibility of a virus toanti-viral drugs known to one of skill in the art can be adapted todetermine replication capacity or to determine antiviral drugsusceptibility or resistance. See, e.g., Shi and Mellors, 1997,Antimicrob Agents Chemother. 41(12):2781-85; Gervaix et al., 1997, ProcNatl Acad Sci U S. A. 94(9):4653-8; Race et al., 1999, AIDS13:2061-2068, incorporated herein by reference in their entireties.

In addition, the phenotypic assays described above can also be used todetermine the effectiveness of candidate compounds. Generally, suchmethods comprise performing the phenotypic assay in the presence andabsence of the candidate compound, wherein the difference in activity orexpression of the indicator gene indicates the effectiveness of thecandidate compound. Advantageously, the methods can be performed in thepresence of a mutation associated with NNRTI resistance as disclosedherein. By performing such assays in the presence of such mutations,candidate compounds can be identified that have beneficial interactionswith the NNRTIs to which the virus is hyper susceptible. In certainembodiments, the candidate compound will have an additive effect onviral inhibition with the NNRTI. In preferred embodiments, the candidatecompound will have a synergistic effect on viral inhibition with theNNRTI. Further guidance may be found in performing the assays todetermine the effectiveness of candidate compounds in Petropoulos etal., 2000, Antimicrob. Agents Chemother. 44:920-928; and U.S. Pat. Nos.5,837,464 and 6,242,187.

5.4.1 Detecting the Presence or Absence of Mutations in a Virus

The presence or absence of an mutation associated with NRTI, NNRTI, orINSTI resistance or susceptibility according to the present invention ina virus can be determined by any means known in the art for detecting amutation. The mutation can be detected in the viral gene that encodes aparticular protein, or in the protein itself, i.e., in the amino acidsequence of the protein.

In one embodiment, the mutation is in the viral genome. Such a mutationcan be in, for example, a gene encoding a viral protein, in a geneticelement such as a cis or trans acting regulatory sequence of a geneencoding a viral protein, an intergenic sequence, or an intron sequence.The mutation can affect any aspect of the structure, function,replication or environment of the virus that changes its resistance toan anti-viral treatment and/or its replication capacity. In oneembodiment, the mutation is in a gene encoding a viral protein that isthe target of an currently available anti-viral treatment. In otherembodiments, the mutation is in a gene or other genetic element that isnot the target of a currently-available anti-viral treatment.

A mutation within a viral gene can be detected by utilizing any suitabletechnique known to one of skill in the art without limitation. Viral DNAor RNA can be used as the starting point for such assay techniques, andmay be isolated according to standard procedures which are well known tothose of skill in the art.

The detection of a mutation in specific nucleic acid sequences, such asin a particular region of a viral gene, can be accomplished by a varietyof methods including, but not limited to,restriction-fragment-length-polymorphism detection based onallele-specific restriction-endonuclease cleavage (Kan and Dozy, 1978,Lancet ii:910-912), mismatch-repair detection (Faham and Cox, 1995,Genome Res 5:474-482), binding of MutS protein (Wagner et aL, 1995, NuclAcids Res 23:3944-3948), denaturing-gradient gel electrophoresis (Fisheret al., 1983, Proc. Natl. Acad. Sci. US.A. 80:1579-83),single-strand-conformation-polymorphism detection (Orita et al., 1983,Genomics 5:874-879), RNAase cleavage at mismatched base-pairs (Myers etal., 1985, Science 230:1242), chemical (Cotton et at, 1988, Proc. Natl.Acad. Sci. U.S.A. 85:4397-4401) or enzymatic (Youil et al., 1995, Proc.Natl. Acad. Sci. US.A. 92:87-91) cleavage of heteroduplex DNA, methodsbased on oligonucleotide-specific primer extension (Syvanen et ai.,1990, Genomics 8:684-692), genetic bit analysis (Nikiforov et al., 1994,Nucl Acids Res 22:4167-4175), oligonucleotide-ligation assay (Landegrenet al., 1988, Science 241:1077), oligonucleotide-specific ligation chainreaction (“LCR”) (Barrany, 1991, Proc. Natl. Acad. Sci. U.S.A.88:189-193), gap-LCR (Abravaya et al., 1995, Nucl Acids Res 23:675-682),radioactive or fluorescent DNA sequencing using standard procedures wellknown in the art, and peptide nucleic acid (PNA) assays (Drum et al.,1993, Nucl. Acids Res. 21:5332-5356; Thiede et al., 1996, Nucl. AcidsRes. 24:983-984).

In addition, viral DNA or RNA may be used in hybridization oramplification assays to detect abnormalities involving gene structure,including point mutations, insertions, deletions and genomicrearrangements. Such assays may include, but are not limited to,Southern analyses (Southern, 1975, J. Moi. Biol. 98:503-517), singlestranded conformational polymorphism analyses (SSCP) (Orita et al.,1989, Proc. Natl. Acad. Sci. USA 86:2766-2770), and PCR analyses (U.S.Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188; PCRStrategies, 1995 Innis et al. (eds.), Academic Press, Inc.).

Such diagnostic methods for the detection of a gene-specific mutationcan involve for example, contacting and incubating the viral nucleicacids with one or more labeled nucleic acid reagents includingrecombinant DNA molecules, cloned genes or degenerate variants thereof,under conditions favorable for the specific annealing of these reagentsto their complementary sequences. Preferably, the lengths of thesenucleic acid reagents are at least 15 to 30 nucleotides. Afterincubation, all non-annealed nucleic acids are removed from the nucleicacid molecule hybrid. The presence of nucleic acids which havehybridized, if any such molecules exist, is then detected. Using such adetection scheme, the nucleic acid from the virus can be immobilized,for example, to a solid support such as a membrane, or a plastic surfacesuch as that on a microtiter plate or polystyrene beads. In this case,after incubation, non-annealed, labeled nucleic acid reagents of thetype described above are easily removed. Detection of the remaining,annealed, labeled nucleic acid reagents is accomplished using standardtechniques well-known to those in the art. The gene sequences to whichthe nucleic acid reagents have annealed can be compared to the annealingpattern expected from a normal gene sequence in order to determinewhether a gene mutation is present.

These techniques can easily be adapted to provide high-throughputmethods for detecting mutations in viral genomes. For example, a genearray from Affymetrix (Affymetrix, Inc., Sunnyvale, Calif.) can be usedto rapidly identify genotypes of a large number of individual viruses.Affymetrix gene arrays, and methods of making and using such arrays, aredescribed in, for example, U.S. Pat. Nos. 6,551,784, 6,548,257,6,505,125, 6,489,114, 6,451,536, 6,410,229, 6,391,550, 6,379,895,6,355,432, 6,342,355, 6,333,155, 6,308,170, 6,291,183, 6,287,850,6,261,776, 6,225,625, 6,197,506, 6,168,948, 6,156,501, 6,141,096,6,040,138, 6,022,963, 5,919,523, 5,837,832, 5,744,305, 5,834,758, and5,631,734, each of which is hereby incorporated by reference in itsentirety.

In addition, Ausubel et al., eds., Current Protocols in MolecularBiology, 2002, Vol. 4, Unit 25B, Ch. 22, which is hereby incorporated byreference in its entirety, provides further guidance on construction anduse of a gene array for determining the genotypes of a large number ofviral isolates. Finally, U.S. Pat. Nos. 6,670,124; 6,617,112; 6,309,823;6,284,465; and 5,723,320, each of which is incorporated by reference inits entirety, describe related array tectmoiogles that can reacmy beadapted for rapid identification of a large number of viral genotypes byone of skill in the art.

Alternative diagnostic methods for the detection of gene specificnucleic acid molecules may involve their amplification, e.g., by PCR(U.S. Pat. Nos. 4,683,202; 4,683,195; 4,800,159; and 4,965,188; PCRStrategies, 1995 Innis et al. (eds.), Academic Press, Inc.), followed bythe detection of the amplified molecules using techniques well known tothose of skill in the art. The resulting amplified sequences can becompared to those which would be expected if the nucleic acid beingamplified contained only normal copies of the respective gene in orderto determine whether a gene mutation exists.

Additionally, the nucleic acid can be sequenced by any sequencing methodknown in the art. For example, the viral DNA can be sequenced by thedideoxy method of Sanger et al., 1977, Proc. Natl. Acad. Sci. USA74:5463, as further described by Messing et al., 1981, Nuc. Acids Res.9:309, or by the method of Maxam et al., 1980, Methods in Enzymology65:499. See also the techniques described in Sambrook et al., 2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,3^(rd) ed., NY; and Ausubel et al., 1989, Current Protocols in MolecularBiology, Greene Publishing Associates and Wiley Interscience, NY.

Antibodies directed against the viral gene products, i.e., viralproteins or viral peptide fragments can also be used to detect mutationsin the viral proteins. Alternatively, the viral protein or peptidefragments of interest can be sequenced by any sequencing method known inthe art in order to yield the amino acid sequence of the protein ofinterest. An example of such a method is the Edman degradation methodwhich can be used to sequence small proteins or polypeptides. Largerproteins can be initially cleaved by chemical or enzymatic reagentsknown in the art, for example, cyanogen bromide, hydroxylamine, trypsinor chymotrypsin, and then sequenced by the Edman degradation method.

5.4.2 Correlating Mutations with Altered Susceptibility to an NRTI,NNRTI, or INSTI

Any method known in the art can be used to determine whether a mutationis correlated with altered susceptibility to an NRTI, NNRTI, or INSTI.In one embodiment, univariate analysis is used to identify mutationscorrelated with altered susceptibility to an NRTI, NNRTI, or INSTI.Univariate analysis yields P values that indicate the statisticalsignificance of the correlation. In such embodiments, the smaller the Pvalue, the more significant the measurement. Preferably the P valueswill be less than 0.05. More preferably, P values will be less than0.01. Even more preferably, the P value will be less than 0.005. Pvalues can be calculated by any means known to one of skill in the art.In one embodiment, P values are calculated using Fisher's Exact Test. Inanother embodiment, P values can be calculated with Student's t-test.See, e.g., David Freedman, Robert Pisani & Roger Purves, 1980,STATISTICS, W. W. Norton, New York. In certain embodiments, P values canbe calculated with both Fisher's Exact Test and Student's t-test. Insuch embodiments, P values calculated with both tests are preferablyless than 0.05. However, a correlation with a P value that is less than0.10 in one test but less than 0.05 in another test can still beconsidered to be a marginally significant correlation. Such mutationsare suitable for further analysis with, for example, multivariateanalysis. Alternatively, further univariate analysis can be performed ona larger sample set to confirm the significance of the correlation.

Further, an odds ratio can be calculated to determine whether a mutationcorrelates with altered susceptibility to an NRTI, NNRTI, or INSTI.Generally, calculation of odds rations depends on dividing thepercentage of virus that comprise a particular mutation or mutationsthat are identified as having altered susceptibility to an NRTI, NNRTI,or INSTI by the percentage of virus with the same mutation or mutationsthat are identified as not having altered susceptibility to an NRTI,NNRTI, or INSTI. In certain embodiments, an odds ratio that is greaterthan one indicates that the mutation does not correlate with alteredsusceptibility to an NRTI, NNRTI, or INSTI. In certain embodiments, anodds ratio that is greater than one indicates that the mutationcorrelates with altered susceptibility to an NRTI, NNRTI, or INSTI.

In yet another embodiment, multivariate analysis can be used todetermine whether a mutation correlates with altered susceptibility toan NRTI, NNRTI, or INSTI. Any multivariate analysis known by one ofskill in the art to be useful in calculating such a correlation can beused, without limitation. In certain embodiments, a statisticallysignificant number of virus's resistance or susceptibility phenotypes,e.g., IC₅0, can be determined. These IC50 values can then be dividedinto groups that correspond to percentiles of the set of IC50 valuesobserved.

After assigning each virus's IC50 value to the appropriate group, thegenotype of that virus can be assigned to that group. By performing thismethod for all viral isolates, the number of instances of a particularmutation in a given percentile of NRTI, NNRTI, or INSTI resistance orsusceptibility can be observed. This allows the skilled practitioner toidentify mutations that correlate with altered susceptibility to anNRTI, NNRTI, or INSTI.

Finally, in yet another embodiment, regression analysis can be performedto identify mutations that best predict NRTI, NNRTI, or INSTI resistanceor susceptibility. In such embodiments, regression analysis is performedon a statistically significant number of viral isolates for whichgenotypes and NRTI, NNRTI, or INSTI resistance or susceptibilityphenotypes have been determined. The analysis then identifies whichmutations appear to best predict, e.g., most strongly correlate withaltered susceptibility to an NRTI, NNRTI, or INSTI. Such analysis canthen be used to construct rules for predicting altered susceptibility toan NRTI, NNRTI, or INSTI based upon knowledge of the genotype of aparticular virus, described below. In certain embodiments, software suchas, for example, CART 5.0, Prism 4.0, or Insightful Miner 3.0 can beused to perform the analysis that identifies the mutations that appearto best predict altered susceptibility to an NRTI, NNRTI, or INSTI.

5.4.3 Computer-Implemented Methods for Determining AlteredSusceptibility to an NRTI, NNRTI, or INSTI and Related Articles

In another aspect, the present invention provides computer-implementedmethods for determining whether an HIV-1 has altered susceptibility toan NRTI, NNRTI, or INSTI. In such embodiments, the methods of theinvention are adapted to take advantage of the processing power ofmodern computers. One of skill in the art can readily adapt the methodsin such a manner.

Therefore, in certain embodiments, the invention provides acomputer-implemented method for determining that an HIV-1 is resistantto an NNRTI or AZT, comprising inputting genetic information into amemory system of a computer, wherein the genetic information indicatesthat the HIV-1 has a gene encoding reverse transcriptase with a mutationat codon 348 or 369, inputting a correlation between the presence of themutations and resistance to an NNRTI or AZT into the memory system ofthe computer, and determining that the HIV-1 is resistant to the NNRTIor AZT. In certain embodiments, the genetic information indicates thatthe mutation at codon 348 encodes isoleucine (I). In certainembodiments, the genetic information indicates that the mutation atcodon 369 encodes isoleucine (I) or alanine (A). In certain embodiments,the NNRTI is EFV, DLV, or NVP. In certain embodiments, the NNRTI is EFV.In certain embodiments, the NNRTI is DLV. In certain embodiments, theNNRTI is NVP. In certain embodiments, the HIV-1 is determined to beresistant to AZT.

In certain embodiments, the genetic information further indicates that amutation at codon 103, 179, 190, or 225 is present in the gene encodingreverse transcriptase in addition to the mutation in codon 348 or 369.In certain embodiments, the genetic information indicates that amutation at codon 103 is present. In certain embodiments, the geneticinformation indicates that the mutation at codon 103 encodes asparagine(N), arginine (R), serine (S), glutamine (Q), or threonine (T). Incertain embodiments, the genetic information indicates that a mutationat codon 225 is present. In certain embodiments, the genetic informationindicates that the mutation at codon 225 encodes histidine (H). Incertain embodiments, the genetic information indicates that a mutationat codon 103 and a mutation at codon 225 are present. In certainembodiments, the genetic information indicates that the mutation atcodon 103 encodes asparagine (N), arginine (R), serine (S), glutamine(Q), or threonine (T). In certain embodiments, the genetic informationindicates that the mutation at codon 225 encodes histidine (H). Incertain embodiments, a mutation at codon 190 is present. In certainembodiments, the genetic information indicates that the mutation atcodon 190 encodes serine (S). In certain embodiments, the geneticinformation indicates that mutations at codon 103 and codon 179 arepresent. In certain embodiments, the genetic information indicates thatthe mutation at codon 103 encodes asparagine (N), arginine (R), serine(S), glutamine (Q), or threonine (T). In certain embodiments, thegenetic information indicates that the mutation at codon 179 encodesaspartic acid (D). In certain embodiments, the NNRTI is EFV, NVP, orDLV. In certain embodiments, the NNRTI is EFV. In certain embodiments,the NNRTI is NVP. In certain embodiments, the NNRTI is DLV.

In another aspect, the invention provides a computer-implemented methodfor determining that an HIV-1 is resistant to an NNRTI, comprisinginputting genetic information into a memory system of a computer,wherein the genetic information indicates that the HIV-1 has a geneencoding reverse transcriptase with a mutation at codon 399 incombination with a mutation at codon 103, 179, or 190, inputting acorrelation between the presence of the mutations and resistance to anNNRTI into the memory system of the computer, and determining that theHIV-1 is resistant to the NNRTI. In certain embodiments, the mutation atcodon 399 encodes aspartic acid (D). In certain embodiments, the geneticinformation indicates that a mutation at codon 103 is present. Incertain embodiments, the genetic information indicates that the mutationat codon 103 encodes asparagine (N), arginine (R), serine (S), glutamine(Q), or threonine (T). In certain embodiments, the genetic informationindicates that a mutation at codon 179 is present. In certainembodiments, the genetic information indicates that the mutation atcodon 179 encodes aspartic acid (D). In certain embodiments, the geneticinformation indicates that a mutation at codon 190 is present. Incertain embodiments, the genetic information indicates that the mutationat codon 190 encodes serine (S). In certain embodiments, the geneticinformation indicates that mutations at codons 103 and 179 are present.In certain embodiments, the genetic information indicates that themutation at codon 103 encodes asparagine (N), arginine (R), serine (S),glutamine (Q), or threonine (T). In certain embodiments, the geneticinformation indicates that the mutation at codon 179 encodes asparticacid (D). In certain embodiments, the NNRTI is EFV, NVP, or DLV. Incertain embodiments, the NNRTI is EFV. In certain embodiments, the NNRTIis NVP. In certain embodiments, the NNRTI is DLV.

In another aspect, the invention provides a computer-implemented methodfor determining that an HIV-1 has reduced replication capacity relativeto a reference HIV-1, comprising inputting genetic information into amemory system of a computer, wherein the genetic information indicatesthat the HIV-1 has a gene encoding reverse transcriptase with a mutationat codon 369 or a gene encoding integrase with a mutation at codon 97,inputting a correlation between the presence of the mutations andreduced replication capacity into the memory system of the computer, anddetermining that the HIV-1 has reduced replication capacity relative toa reference HIV. In certain embodiments, the genetic informationindicates that a mutation at codon 369 of reverse transcriptase ispresent. In certain embodiments, the genetic information indicates thatthe mutation at codon 369 encodes isoleucine (I). In certainembodiments, the genetic information indicates that a mutation at codon97 of integrase is present. In certain embodiments, the geneticinformation indicates that the mutation at codon 97 encodes alanine (A).In certain embodiments, the reference HIV-1 is NL4-3. In certainembodiments, the reference HIV-1 has an identical genotype to the HIV-1having its replication capacity determined except for codon 369 ofreverse transcriptase or codon 97 of integrase. In certain embodiments,the genotype of the reference HIV-1 differs from the HIV-1 having itsreplication capacity determined at codon 369 of reverse transcriptase.In certain embodiments, the genotype of the reference HIV-1 differs fromthe HIV-1 having its replication capacity determined at codon 97 ofintegrase.

In another aspect, the invention provides a computer-implemented methodfor determining that an HIV-1 has altered susceptibility to a integrasestrand transfer inhibitor (INSTI), comprising inputting geneticinformation into a memory system of a computer, wherein the geneticinformation indicates that the HIV-1 has a gene encoding integrase witha mutation at codon 97 or 156, inputting a correlation between thepresence of the mutations and altered susceptibility to an INSTI intothe memory system of the computer, and determining that the HIV-1 hasaltered susceptibility to the INSTI. In certain embodiments, the HIV-1exhibits increased susceptibility to the INSTI. In certain embodiments,the HIV-1 exhibits decreased susceptibility to the INSTI. In certainembodiments, the genetic information indicates that a mutation at codon97 is present. In certain embodiments, the genetic information indicatesthat the mutation at codon 97 encodes alanine (A). In certainembodiments, the genetic information indicates that a mutation at codon156 is present. In certain embodiments, the genetic informationindicates that the mutation at codon 156 encodes asparagines (N). Incertain embodiments, the INSTI is a napthyridine carboximide. In certainembodiments, the INSTI is L-870,810.

In certain embodiments, the genetic information further indicates amutation at codon 66, 72, 121, 125, 154, or 155 is present in the geneencoding integrase. In certain embodiments, the HIV-1 exhibits decreasedsusceptibility to the INSTI. In certain embodiments, the geneticinformation indicates that a mutation at codon 66 is present. In certainembodiments, the genetic information indicates that the mutation atcodon 66 encodes isoleucine (I). In certain embodiments, the geneticinformation indicates that a mutation at codon 72 is present. In certainembodiments, the genetic information indicates that the mutation atcodon 72 encodes isoleucine (I). In certain embodiments, the geneticinformation indicates that a mutation at codon 121 is present. Incertain embodiments, the genetic information indicates that the mutationat codon 121 encodes tyrosine (Y). In certain embodiments, the geneticinformation indicates that a mutation at codon 125 is present. Incertain embodiments, the genetic information indicates that the mutationat codon 125 encodes lysine (K). In certain embodiments, the geneticinformation indicates that a mutation at codon 154 is present. Incertain embodiments, the genetic information indicates that the mutationat codon 154 encodes isoleucine (I). In certain embodiments, the geneticinformation indicates that a mutation at codon 155 is present. Incertain embodiments, the mutation at codon 155 encodes serine (S). Incertain embodiments, the genetic information indicates that mutations atcodons 72, 121, and 125 are present. In certain embodiments, the geneticinformation indicates that the mutation at codon 72 encodes isoleucine(I), the mutation at codon 121 encodes tyrosine (Y), and the mutation atcodon 125 encodes lysine (K). In certain embodiments, the geneticinformation indicates that mutations at codons 66 and 154 are present.In certain embodiments, the genetic information indicates that themutation at codon 66 encodes isoleucine (I) and the mutation at codon154 encodes isoleucine (I). In certain embodiments, the INSTI is adiketo acid. In certain embodiments, the INSTI is diketo acid 1 ordiketo acid 2. In certain embodiments, the INSTI is a napthyridinecarboximide. In certain embodiments, the INSTI is L-870,810.

In certain embodiments, the methods further comprise displaying that theHIV-1 has altered susceptibility to an NRTI, NNRTI, or INSTI on adisplay of the computer. In certain embodiments, the methods furthercomprise printing that the HIV-1 has altered susceptibility to an NRTI,NNRTI, or INSTI. In certain embodiments, the methods further comprisedisplaying that the HIV-1 has increased susceptibility to an NRTI,NNRTI, or INSTI on a display of the computer. In certain embodiments,the methods further comprise printing that the HIV-1 has increasedsusceptibility to an NRTI, NNRTI, or INSTI. In certain embodiments, themethods further comprise displaying that the HIV-1 has decreasedsusceptibility to an NRTI, NNRTI, or INSTI on a display of the computer.In certain embodiments, the methods further comprise printing that theHIV-1 has decreased susceptibility to an NRTI, NNRTI, or INSTI.

In another aspect, the invention provides a tangible medium comprisingdata indicating that an HIV-1 has altered susceptibility to an NRTI,NNRTI, or INSTI because of the presence of one or more mutationscorrelated with altered susceptibility to an NRTI, NNRTI, or INSTI asdisclosed herein. In certain embodiments, the tangible medium is a paperdocument indicating that an HIV-1 is has altered susceptibility to anNRTI, NNRTI, or INSTI. In certain embodiments, the paper document is aprinted document, e.g., a computer print-out. In certain embodiments,the tangible medium is a computer-readable medium comprising dataindicating that an HIV-1 is has altered susceptibility to an NRTI,NNRTI, or INSTI. In certain embodiments, the computer-readable medium isa random-access memory. In certain embodiments, the computer-readablemedium is a fixed disk. In certain embodiments, the computer-readablemedium is a floppy disk. In certain embodiments, the computer-readablemedium is a portable memory device, such as, e.g., a USB key or anIPOD®. In certain embodiments, the HIV-1 with altered susceptibility toan NRTI, NNRTI, or INSTI has reduced susceptibility. In certainembodiments, the HIV-1 with altered susceptibility to an NRTI, NNRTI, orINSTI has increased susceptibility.

In still another aspect, the invention provides an article ofmanufacture that comprises computer-readable instructions for performinga method of the invention. In certain embodiments, the article ofmanufacture is a random-access memory. In certain embodiments, thearticle of manufacture is a fixed disk. In certain embodiments, thearticle of manufacture is a floppy disk. In certain embodiments, thearticle of manufacture is a portable memory device, such as, e.g., a USBkey or an IPOD®.

In yet another aspect, the invention provides a computer-readable mediumthat comprises data that that can be utilized in conjunction with amethod to determine whether an HIV-1 and computer-readable instructionsfor performing a method of the invention. In certain embodiments, thecomputer-readable medium is a random-access memory. In certainembodiments, the computer-readable medium is a fixed disk. In certainembodiments, the computer-readable medium is a floppy disk. In certainembodiments, the computer-readable medium is a portable memory device,such as, e.g., a USB key or an IPOD®.

In yet another aspect, the invention provides a computer system that isconfigured to perform a method of the invention.

5.4.4 Viruses and Viral Samples

A mutation associated with altered susceptibility to an NRTI, NNRTI, orINSTI according to the present invention can be present in any type ofvirus. For example, such mutations may be identified in any virus thatinfects animals known to one of skill in the art without limitation. Inone embodiment of the invention, the virus includes viruses known toinfect mammals, including dogs, cats, horses, sheep, cows etc. Incertain embodiment, the virus is known to infect primates. In preferredembodiments, the virus is known to infect humans. Examples of suchviruses that infect humans include, but are not limited to, humanimmunodeficiency virus (“HIV”), herpes simplex virus, cytomegalovirusvirus, varicella zoster virus, other human herpes viruses, influenza A,B and C virus, respiratory syncytial virus, hepatitis A, B and Cviruses, rhinovirus, and human papilloma virus. In certain embodiments,the virus is HCV. In other embodiments, the virus is HBV. In a preferredembodiment of the invention, the virus is HIV. Even more preferably, thevirus is human immunodeficiency virus type 1 (“HIV-1”). The foregoingare representative of certain viruses for which there is presentlyavailable anti-viral chemotherapy and represent the viral familiesretroviridae, herpesviridae, orthomyxoviridae, paramxyxoviridae,picornaviridae, flaviviridae, pneumoviridae and hepadnaviridae. Thisinvention can be used with other viral intections clue to other viruseswithin these families as well as viral infections arising from virusesin other viral families for which there is or there is not a currentlyavailable therapy.

A mutation associated with altered susceptibility to an NRTI, NNRTI, orINSTI according to the present invention can be found in a viral sampleobtained by any means known in the art for obtaining viral samples. Suchmethods include, but are not limited to, obtaining a viral sample from ahuman or an animal infected with the virus or obtaining a viral samplefrom a viral culture. In one embodiment, the viral sample is obtainedfrom a human individual infected with the virus. The viral sample couldbe obtained from any part of the infected individual's body or anysecretion expected to contain the virus. Examples of such parts include,but are not limited to blood, serum, plasma, sputum, lymphatic fluid,semen, vaginal mucus and samples of other bodily fluids. In a preferredembodiment, the sample is a blood, serum or plasma sample.

In another embodiment, a mutation associated with altered susceptibilityto an NRTI, NNRTI, or INSTI according to the present invention ispresent in a virus that can be obtained from a culture. In someembodiments, the culture can be obtained from a laboratory. In otherembodiments, the culture can be obtained from a collection, for example,the American Type Culture Collection.

In certain embodiments, a mutation associated with alteredsusceptibility to an NRTI, NNRTI, or INSTI according to the presentinvention is present in a derivative of a virus. In one embodiment, thederivative of the virus is not itself pathogenic. In another embodiment,the derivative of the virus is a plasmid-based system, whereinreplication of the plasmid or of a cell transfected with the plasmid isaffected by the presence or absence of the selective pressure, such thatmutations are selected that increase resistance to the selectivepressure. In some embodiments, the derivative of the virus comprises thenucleic acids or proteins of interest, for example, those nucleic acidsor proteins to be targeted by an anti-viral treatment. In oneembodiment, the genes of interest can be incorporated into a vector.See, e.g., U.S. Pat. Nos. 5,837,464 and 6,242,187 and PCT publication,WO 99/67427, each of which is incorporated herein by reference. Incertain embodiments, the genes can be those that encode for a proteaseor reverse transcriptase.

In another embodiment, the intact virus need not be used. Instead, apart of the virus incorporated into a vector can be used. Preferablythat part of the virus is used that is targeted by an anti-viral drug.

In another embodiment, a mutation associated with altered susceptibilityto an NRTI, NNRTI, or INSTI according to the present invention ispresent in a genetically modified virus. The virus can be geneticallymodified using any method known in the art for genetically modifying avirus. For example, the virus can be grown for a desired number ofgenerations in a laboratory culture. In one embodiment, no selectivepressure is applied (i.e., the virus is not subjected to a treatmentthat favors the replication of viruses with certain characteristics),and new mutations accumulate through random genetic drift. In anotherembodiment, a selective pressure is applied to the virus as it is grownin culture (i.e., the virus is grown under conditions that favor thereplication of viruses having one or more characteristics). In oneembodiment, the selective pressure is an anti-viral treatment. Any knownanti-viral treatment can be used as the selective pressure.

In certain embodiments, the virus is HIV and the selective pressure isan NNRTI. In another embodiment, the virus is HIV-1 and the selectivepressure is an NNRTI. Any NNRTI can be used to apply the selectivepressure. Examples of NNRTIs include, but are not limited to,nevirapine, delavirdine and efavirenz. By treating HIV cultured in vitrowith an NNRTI, one can select for mutant strains of HIV that have anincreased resistance to the NNRTI. The stringency of the selectivepressure can be manipulated to increase or decrease the survival ofviruses not having the selected-for characteristic.

In other embodiments, the virus is HIV and the selective pressure is aNRTI. In another embodiment, the virus is HIV-1 and the selectivepressure is a NRTI. Any NRTI can be used to apply the selectivepressure. Examples of NRTIs include, but are not limited to, AZT, ddl,ddC, d4T, 3TC, abacavir, and tenofovir. By treating HIV cultured invitro with a NRTI, one can select for mutant strains of HIV that have anincreased resistance to the NRTI. The stringency of the selectivepressure can be manipulated to increase or decrease the survival ofviruses not having the selected-for characteristic.

In still other embodiments, the virus is HIV and the selective pressureis a PI. In another embodiment, the virus is HIV-1 and the selectivepressure is a PI. Any PI can be used to apply the selective pressure.Examples of PIs include, but are not limited to, saquinavir, ritonavir;inctinavir, neltinavir, amprenavir, lopinavir and atazanavir. Bytreating HIV cultured in vitro with a PI, one can select for mutantstrains of HIV that have an increased resistance to the PI. Thestringency of the selective pressure can be manipulated to increase ordecrease the survival of viruses not having the selected-forcharacteristic.

In still other embodiments, the virus is HIV and the selective pressureis an entry inhibitor. In another embodiment, the virus is HIV-1 and theselective pressure is an entry inhibitor. Any entry inhibitor can beused to apply the selective pressure. An example of a entry inhibitorincludes, but is not limited to, fusion inhibitors such as, for example,enfuvirtide. Other entry inhibitors include co-receptor inhibitors, suchas, for example, AMD3100 (Anormed). Such co-receptor inhibitors caninclude any compound that interferes with an interaction between HIV anda co-receptor, e.g., CCR5 or CRCX4, without limitation. By treating HIVcultured in vitro with an entry inhibitor, one can select for mutantstrains of HIV that have an increased resistance to the entry inhibitor.The stringency of the selective pressure can be manipulated to increaseor decrease the survival of viruses not having the selected-forcharacteristic.

In still other embodiments, the virus is HIV and the selective pressureis an INSTI. In another embodiment, the virus is HIV-1 and the selectivepressure is an INSTI. Any INSTI can be used to apply the selectivepressure. Examples of INSTIs include, but are not limited to, diketoacid 1, diketo acid 2, and L-870,810. By treating HIV cultured in vitrowith an INSTI, one can select for mutant strains of HIV that have anincreased resistance to the INSTI. The stringency of the selectivepressure can be manipulated to increase or decrease the survival ofviruses not having the selected-for characteristic.

In another aspect, a mutation associated with altered susceptibility toan NRTI, NNRTI, or INSTI according to the present invention can be madeby mutagenizing a virus, a viral genome, or a part of a viral genome.Any method of mutagenesis known in the art can be used for this purpose.In certain embodiments, the mutagenesis is essentially random. Incertain embodiments, the essentially random mutagenesis is performed byexposing the virus, viral genome or part of the viral genome to amutagenic treatment. In another embodiment, a gene that encodes a viralprotein that is the target of an anti-viral therapy is mutagenized.Examples of essentially random mutagenic treatments include, forexample, exposure to mutagenic substances (e.g., ethidium bromide,ethylmethanesulphonate, ethyl nitroso urea (ENU) etc.) radiation (e.g.,ultraviolet light), the insertion and/or removal of transposableelements (e.g., Tn5, Tn10), or replication in a cell, cell extract, orin vitro replication system that has an increased rate of mutagenesis.See, e.g., Russell et al., 1979, Proc. Nat. Acad. Sci. USA 76:5918-5922;Russell, W., 1982, Environmental Mutagens and Carcinogens: Proceedingsof the Third International Conference on Environmental Mutagens. One ofskill in the art will appreciate that while each of these methods ofmutagenesis is essentially random, at a molecular level, each has itsown preferred targets.

In another aspect, a mutation associated with altered susceptibility toan NRTI, NNRTI, or INSTI can be made using site-directed mutagenesis.Any method of site-directed mutagenesis known in the art can be used(see e.g., Sambrook et al., 2001, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, 3^(rd) ed., NY; and Ausubel etal., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, NY). See, e.g., Sarkar and Sommer,1990, Biotechniques, 8:404-407. The site directed mutagenesis can bedirected to, e.g., a particular gene or genomic region, a particularpart of a gene or genomic region, or one or a few particular nucleotideswithin a gene or genomic region. In one embodiment, the site directedmutagenesis is directed to a viral genomic region, gene, gene fragment,or nucleotide based on one or more criteria. In one embodiment, a geneor a portion of a gene is subjected to site-directed mutagenesis becauseit encodes a protein that is known or suspected to be a target of ananti-viral therapy, e.g., the gene encoding the HIV reversetranscriptase. In another embodiment, a portion of a gene, or one or afew nucleotides within a gene, are selected for site-directedmutagenesis. In one embodiment, the nucleotides to be mutagenized encodeamino acid residues that are known or suspected to interact with ananti-viral compound. In another embodiment, the nucleotides to bemutagenized encode amino acid residues that are known or suspected to bemutated in viral strains that are resistant or susceptible orhypersusceptible to one or more antiviral agents. In another embodiment,the mutagenized nucleotides encode amino acid residues that are adjacentto or near in the primary sequence of the protein residues known orsuspected to interact with an anti-viral compound or known or suspectedto be mutated in viral strains that are resistant or susceptible orhypersusceptible to one or more antiviral agents. In another embodiment,the mutagenized nucleotides encode amino acid residues that are adjacentto or near to in the secondary, tertiary or quaternary structure of theprotein residues known or suspected to interact with an anti-viralcompound or known or suspected to be mutated in viral strains having analtered replication capacity. In another embodiment, the mutagenizednucleotides encode amino acid residues in or near the active site of aprotein that is known or suspected to bind to an anti-viral compound.

6. EXAMPLES 6.1 Example 1 Measuring NRTI or NNRTI Resistance UsingVectors Comprising Patient Derived Segments Corresponding to the HIVProtease and Reverse Transcriptase (PR-RT) Coding Regions

This example provides methods and compositions for accurately andreproducibly measuring the resistance or sensitivity of HIV-1 toantiretroviral drugs including, for example, NRTIs such as AZT andNNRTIs such as EFV, DLV, and/or NVP. The methods for measuringresistance or susceptibility to such drugs can be adapted to other HIVstrains, such as HIV-2, or to other viruses, including, but not limitedto hepadnaviruses (e.g., human hepatitis B virus), flaviviruses (e.g.,human hepatitis C virus) and herpesviruses (e.g., humancytomegalovirus).

Drug resistance tests can be carried out, for example, using the methodsfor phenotypic drug susceptibility and resistance tests described inU.S. Pat. No. 5,837,464 (International Publication Number WO 97/27319)which is hereby incorporated by reference in its entirety, or accordingto the protocol that follows.

Patient-derived segments corresponding to the HIV protease and reversetranscriptase coding regions (hereinafter “PR-RT) were amplified by thereverse transcription-polymerase chain reaction method (RT-PCR) usingviral RNA isolated from viral particles present in the plasma or serumof HIV-infected individuals as follows. Viral RNA was isolated from theplasma or serum using oligo-dT magnetic beads (Dynal Biotech, Oslo,Norway), followed by washing and elution of viral RNA. The RT-PCRprotocol was divided into two steps. A retroviral reverse transcriptase(e.g. Moloney MuLV reverse transcriptase (Roche Molecular Systems, Inc.,Branchburg, N.J.; Invitrogen, Carlsbad, Calif.), or avian myeloblastosisvirus (AMV) reverse transcriptase, (Boehringer Mannheim, Indianapolis,Ind.)) was used to copy viral RNA into cDNA. The cDNA was then amplifiedusing a thermostable DNA polymerase (e.g. Taq (Roche Molecular Systems,Inc., Branchburg, N.J.), Tth (Roche Molecular Systems, Inc., Branchburg,N.J.), PRIMEZYME. (enzyme_isolated from Thermus brockianus, Biometra,Gottingen, Germany)) or a combination of thermostable polymerases asdescribed for the performance of “long PCR” (Barnes, W. M., 1994, Proc.Natl. Acad. Sci, USA 91, 2216-20) (e.g. Expand High Fidelity PCR System(Taq+Pwo), (Boehringer Mannheim. Indianapolis, Ind.); GENEAMP® XL PCRkit (PCR kit using_Tth+Vent), (Roche Molecular Systems, Inc.,Branchburg, N.J.); or ADVANTAGE II®, Clontech, Palo Alto, Calif.)

PCR primers were designed to introduce ApaI and PinAl recognition sitesinto the 5′ or 3′ end of the PCR product, respectively.

Resistance test vectors incorporating the “test” patient-derivedsegments were constructed as described in U.S. Pat. No. 5,837,464 usingan amplified DNA product of 1.5 kB prepared by RT-PCR using viral RNA asa template and oligonucleotides PDS Apa, PDS Age, PDS PCR6, Apa-gen,Apa-c, Apa-f, Age-gen, Age-a, RT-ad, RT-b, RT-c, RT-f, and/or RT-g asprimers, followed by digestion with ApaI and Agel or the isoschizomerPinAl. To ensure that the plasmid DNA corresponding to the resultantfitness test vector comprises a representative sample of the HIV viralquasi-species present in the serum of a given patient, many (>250)independent E. coli transformants obtained in the construction of agiven fitness test vector were pooled and used for the preparation ofplasmid DNA.

A packaging expression vector encoding an amphotrophic MuLV 4070A envgene product enables production in a resistance test vector host cellcomprising the vector alone viral particles which can efficiently infecthuman target cells. Vectors encoding all HIV genes with the exception ofenv were used to transfect a packaging host cell (once transfected thehost cell is referred to as a fitness test vector host cell). Thepackaging expression vector which encodes the amphotrophic MuLV 4070Aenv gene product is used with the resistance test vector to enableproduction in the resistance test vector host cell of infectiouspseudotyped resistance test vector viral particles.

Drug resistance tests performed with resistance test vectors werecarried out using packaging host and target host cells consisting of thehuman embryonic kidney cell line 293.

Resistance tests were carried out with resistance test vectors using twohost cell types. Resistance test vector viral particles were produced bya first host cell (the resistance test vector host cell) that wasprepared by transfecting a packaging host cell with the resistance testvector and the packaging expression vector. The resistance test vectorviral particles were then used to infect a second host cell (the targethost cell) in which the expression of the indicator gene is measured.

The resistance test vectors containing a functional luciferase genecassette were constructed as described above and host cells weretransfected with the resistance test vector DNA. The resistance testvectors contained patient-derived reverse transcriptase and protease DNAsequences that encode proteins which were either susceptible orresistant to the antiretroviral agents, such as, for example, NRTIs,NNRTIs, and PIs.

The amount of luciferase activity detected in infected cells is used asa direct measure of “infectivity,” i.e., the ability of the virus tocomplete a single round of replication. Thus, drug resistance orsensitivity can be determined by plotting the amount of luciferaseactivity produced by patient derived viruses in the presence of varyingconcentrations of the antiviral drug. By identifying the concentrationof drug at which luciferase activity is half-maximum, the IC₅₀ of thevirus from which patient-derived segment(s) were obtained for theantiretroviral agent can be determined.

Host (293) cells were seeded in 10-cm-diameter dishes and weretransfected one day after plating with resistance test vector plasmidDNA and the envelope expression vector. Transfections were performedusing a calcium-phosphate co-precipitation procedure. The cell culturemedia containing the DNA precipitate was replaced with fresh medium,from one to 24 hours, after transfection. Cell culture medium containingresistance test vector viral particles was harvested one to four daysafter transfection and was passed through a 0.45-mm filter beforeoptional storage at −80 ° C. Before infection, target cells (293 cells)were plated in cell culture media. Control infections were performedusing cell culture media from mock transfections (no DNA) ortransfections containing the resistance test vector plasmid DNA withoutthe envelope expression plasmid. One to three or more days afterinfection the media was removed and cell lysis buffer (Promega Corp.;Madison, Wis.) was added to each well. Cell lysates were assayed forluciferase activity. Alternatively, cells were lysed and luciferase wasmeasured by adding Steady-Glo (Promega Corp.; Madison, Wis.) reagentdirectly to each well without aspirating the culture media from thewell. The amount of luciferase activity produced in infected cells wasnormalized to adjust for variation in transfection efficiency in thetransfected host cells by measuring the luciferase activity in thetransfected cells, which is not dependent on viral gene functions, andadjusting the luciferase activity from infected cell accordingly.

6.2 Example 2 Measuring NRTI, NNRTI, or INSTI Resistance UsingResistance Test Vectors Comprising Patient Derived SegmentsCorresponding to the Entire pol Gene or a Portion Thereof

This example provides methods and compositions for accurately andreproducibly measuring the resistance or sensitivity of HIV infecting apatient to antiretroviral drugs including. The methods for measuringresistance or susceptibility to such drugs can be adapted to otherviruses, including, but not limited to hepadnaviruses (e.g., humanhepatitis B virus), flaviviruses (e.g., human hepatitis C virus) andherpesviruses (e.g., human cytomegalovirus). The methods described inthis example can also be used to determine the replication capacity ofthe HIV.

The drug resistance tests described herein are a modification of themethods for phenotypic drug susceptibility and resistance testsdescribed in U.S. Pat. No. 5,837,464 (International Publication NumberWO 97/27319) which is hereby incorporated by reference in its entirety.

6.2.1 Construction of Resistance Test Vector Libraries

Patient-derived segment(s) corresponding to the entire pol gene,encoding HIV protease, reverse transcriptase (including RNAse H), andintegrase (hereinafter “POL”), the portion of pol encoding amino acids1-305 of reverse transcriptase (hereinafter “PR-RT”), the portion of polencoding amino acids 319-440 of reverse transcriptase (including RNAseH), and integrase (hereinafter “RHIN”), were amplified by the reversetranscription-polymerase chain reaction method (RT-PCR) using viral RNAisolated from viral particles present in the plasma or serum ofHIV-infected individuals as described below. The portion of pol encodingprotease, amino acids 1-531 of reverse transcriptase (including RNase H)(hereinafter “PRRT-RH”), or the portion of pol encoding protease,reverse transcriptase (including RNAse H) and integrase (hereinafter“PRRT-RHIN”), were assembled from amplicons described above by standardrecombinant DNA techniques involving a KpnI restriction site at aminoacid 400 or a Pin A1 site at amino acid 315.

Virus was pelleted by centrifugation at 20,400×g for 60 min from plasma(typically, 1 ml) prepared from blood samples collected in evacuatedtubes containing either EDTA, acid-citrate dextrose, or heparin as ananticoagulant. Virus particles were disrupted by resuspending thepellets in 200 [1.1 of lysis buffer (4 M guanidine thiocyanate, 0.1 MTris HCl (pH 8.0), 0.5% sodium lauryl sarcosine, 1% dithiothreitol). RNAwas extracted from viral lysates by using oligo(dT) linked to magneticbeads (Dynal, Oslo, Norway). Reverse transcription was performed withSuperscript III (Invitrogen) at 50 degrees for 1 hour using primer 1.All primer sequences are listed in Table 1, below.

TABLE 1 Reverse Transcriptase Primer Gene Primer is Name SEQ ID NO.Located in Sequence Amplicon Primer 1 SEQ ID NO: 1 vif 5′CTTTCCTCGAGAYATACATATGGTGT 3′ POL and RHIN PCR Primers Gene Primer isName Located in Sequence Amplicon Direction Primer 2 SEQ ID NO: 2 pol 5′CAGRGARATTCTAAAAGAACCGGTACATGG 3′ RHIN 5′ Primer 3 SEQ ID NO: 3 gag 5′TTGCAGGGCCCCTAGRAAAAARGGCTG 3′ POL 5′ Primer 4 SEQ ID NO: 4 vif 5′CTTTCCTCGAGAYATACATATGGTGTTTTAC 3′ POL and RHIN 3′

From the resultant cDNA either POL or RHIN sequences were amplifiedusing the Advantage High Fidelity PCR kit (BD Biosciences; Clontech).POL amplification products are made using forward Primer 3 containing anApal site and reverse Primer 4 containing a Xho 1 site. RHINamplification products are made using forward Primer 2 containing aP1NA1 site and reverse Primer 4 containing a Xho 1 site. PCR cyclinginvolves 40 cycles of a 3 step program according to the protocol shownin Table 2, below.

TABLE 2 PCR PROFILE DEGREES MINUTES AMPLIFICATION PROTOCOL FOR RHINDENATURE 94 2:00 40 CYCLES OF: DENATURE 94 0:40 ANNEAL 60 1:00 EXTEND 722:00 EXTENSION 72 10:00  HOLD 4 INDEF AMPLIFICATION OF POL DENATURE 942:00 40 CYCLES OF: DENATURE 94 0:40 ANNEAL 58 1:00 EXTEND 72 3:00EXTENTSION 72 10:00  HOLD 4 INDEF

A retroviral vector designed to measure antiretroviral drugsusceptibility was constructed by using an infectious molecular clone ofHIV-1. The vector, referred to herein as an indicator gene viral vector(IGVV), is replication defective and contains a luciferase expressioncassette inserted within a deleted region of the envelope (env) gene.The IGVV is described in U.S. Pat. No. 5,837,464 (InternationalPublication Number WO 97/27319) which is hereby incorporated byreference in its entirety. This retroviral vector was further modifiedto allow insertion of either the entire pol gene (POL) or the portion ofpoi encoding amino acids 319-440 of reverse transcriptase, the RNase Hportion of reverse transcriptase, and integrase (REIN) by engineering anXho l restriction enzyme recognition site into vif. Prior to doing this,an Xho 1 site in nef was deleted. Resistance Test vectors (RTVs) wereconstructed by incorporating amplified POL or RHIN into the IGVV byusing Apal and Xhol or PinAI and Xho 1 restriction sites respectively.RTVs were prepared as libraries (pools) in order to capture and preservethe pol or RHIN sequence heterogeneity of the virus in the patient. POLamplification products were digested with Apal and Xhol, purified byagarose gel electrophoresis, and ligated to ApaI- and Xhol-digested IGVVDNA. RHIN amplification products were digested with PinAI and Xho/,purified by agarose gel electrophoresis, and ligated to PinAI andXhol-digested IGVV DNA. Ligation reactions were used to transformcompetent Escherichia coli (Invitrogen, Carlsbad, Calif.). An aliquot ofeach transformation was plated onto agar, and colony counts were used toestimate the number of patient-derived segments represented in each RTVlibrary. RTV libraries that comprised less than 50 members are notconsidered representative of the patient virus.

A packaging expression vector encoding an amphotrophic MuLV 4070A envgene product (described in U.S. Pat. No. 5,837,464) enables productionin a host cell of viral particles which can efficiently infect humantarget cells. RTV libraries encoding all HIV genes with the exception ofenv, produced as described above, were used to transfect a packaginghost cell. The packaging expression vector which encodes theamphotrophic MuLV 4070A env gene product is used with the resistancetest vector to enable production of infectious pseudotyped viralparticles comprising the resistance test vector libraries.

6.2.2 Anti-HIV Drug Resistance Assays with Resistance Test VectorsComprising Different POL Sequences

Drug resistance tests performed with test vectors were carried out usingpackaging host and target host cells consisting of the human embryonickidney cell line 293.

Resistance tests were carried out with the RTV libraries by using viralparticles comprising the RTV libraries to infect a host cell in whichthe expression of the indicator gene is measured. The amount ofindicator gene (luciferase) activity detected in infected cells is usedas a direct measure of “infectivity,” i.e., the ability of the virus tocomplete a single round of replication. Thus, drug resistance orsensitivity can be determined by plotting the amount of luciferaseactivity produced by patient derived viruses in the presence of varyingconcentrations of the antiviral drug. By identifying the concentrationof drug at which luciferase activity is half-maximum, the IC50 of thevirus from which patient-derived segment(s) were obtained for theantiretroviral agent can be determined. The IC50 provides a directmeasure of the resistance or susceptibility of the HIV infecting thepatient to the antiviral drug.

In the resistance tests, packaging host (293) cells were seeded in10-cm-diameter dishes and were transfected one day after plating withtest vector plasmid DNA and the envelope expression vector.Transfections were performed using a calcium-phosphate co-precipitationprocedure. The cell culture media containing the DNA precipitate wasreplaced with fresh medium, from one to 24 hours, after transfection.Cell culture medium containing viral particles comprising the TVlibraries was harvested one to four days after transfection and waspassed through a 0.45-mm filter before optional storage at −80 ° C.Before infection, host cells (293 cells) to be infected were plated incell culture media containing varying concentrations of L-870,810, theanti-HIV agent to be tested in the assay. Control infections wereperformed using cell culture media from mock transfections (no DNA) ortransfections containing the test vector plasmid DNA without theenvelope expression plasmid. One to three or more days after infectionthe media was removed and cell lysis buffer (Promega Corp.; Madison,Wis.) was added to each well. Cell lysates were assayed for luciferaseactivity. Alternatively, cells were lysed and luciferase was measured byadding Steady-Glo (Promega Corp.; Madison, Wis.) reagent directly toeach well without aspirating the culture media from the well. The amountof luciferase activity produced in infected cells was normalized toadjust for variation in transfection efficiency in the transfected hostcells by measuring the luciferase activity in the transfected cells,which is not dependent on viral gene functions, and adjusting theluciferase activity from infected cell accordingly. The normalizedluciferase activity was then plotted as a function of the log ofanti-HIV agent present to determine the IC50 of the assayed HIV.

6.2.3 Anti-HIV Drug Resistance Assays Using Different Resistance TestVectors

Different types of resistance test vectors were constructed and used asdescribed in Example 1 and Example 2. As described in Example 1,resistance test vectors comprising patient-derived segmentscorresponding to the HIV-protease and reverse transcriptase codingregion from patient viruses were constructed. Further, as described inExample 2, resistance test vectors comprising patient-derived segmentscorresponding to the entire pol gene, encoding HIV protease, reversetranscriptase (including RNAse H), and integrase (“POL”), and theportion of pol encoding amino acids 1-305 of reverse transcriptase(“PR-RT”), from the above patient viruses were constructed.

Assays using these resistance test vectors were performed for 27different patient samples using the 3 different amplicons to assess theresistance to NNRTIs of HIV isolated from HIV-infected patients. Results(fold change in IC50 or “FC”) for each of these different ampliconsprepared from the same patient samples were compared to assess thedifference.

FIG. 1 shows that overall there are no statistical difference inresistance to three NNRTIs between the resistance test vectorscomprising POL and those comprising PR-RT (student's test, P>0.3).However certain samples displayed lower or higher levels of resistancewhen tested using the POL amplicon compared to the PR-RT amplicon.

6.3 Example 3 Identifying Mutations Correlated with Resistance to anNNRTI

This example provides methods and compositions for identifying mutationsthat correlate with resistance to an NNRT1. Resistance test vectorsderived from patient samples or clones derived from the resistance testvector pools were tested in a resistance assay to determine accuratelyand quantitatively the relative EFV, DLV, or NVP resistance orsusceptibility compared to the median observed resistance orsusceptibility.

6.3.1 Identification of Mutations that Correlate with Resistance to anNNRTI

To identify mutations associated with NNRTI resistance, the drugresistance assays were performed as described in Examples 1 and 2. Inthese assays, POL, PR-RT and RHIN sequences from 27 HIV patient viruseswere successfully amplified and tested for resistance to EFV, NVP andDLV. Viruses that exhibit an 10₅0 2.5 fold higher than wild-type viruswere designated as having reduced susceptibility to the respectiveNNRTI. The results of the assays testing the susceptibility of HIVisolated from 27 HIV-infected patients are presented in Table 3.

As shown in Table 3, nearly all patient samples exhibited consistentfold change values to all three NNRTIs for the three different amplicons(POL, PR-RI and RHIN). However, one patient sample, sample 62, exhibiteddivergent results in the assays using the POL-based resistance testvector from that using the PR-RT-based resistance test vector. Inparticular, sample 62 exhibited 6- to 10-fold higher fold change (“FC”)to NNRTIs when the POL-based resistance test vector were used incomparison to the PR-RT-based resistance test vectors (e.g., EFV FC was202-fold for the POL-based resistance vectors, While EFV FC was only20-fold for the PR-RT-based resistance test vectors).

To further explore this result, resistant test vectors comprisingpatient-derived segments corresponding to different domain of pol genefrom sample 62 were constructed. Diagrammatic representations of theseconstructs are presented as FIG. 2. Resistance assays to NVP using thesedifferent resistance test vectors were performed. FIG. 3 shows NVPresistance results with different amplicons of sample 62. Amplicons ofPRRT-RHIN or PRRT-RH exhibited >200, fold change to NVP, which issimilar to the fold change of amplicon of POL. In contrast, amplicon ofPR-RT exhibited only 64-fold change to NVP. As shown in Table 5, similarresults were observed when resistance assays to DLV and EFV wereperformed using different amplicons of sample 62. These results suggestthat mutations in the C-terminus of reverse transcriptase after codon305 and/or in RNase H are responsible for the observed increase inresistance.

Next, genotypic analysis of patient sample 62 was performed. Patient HIVsample sequences were determined using viral RNA purification, RT/PCRand ABI chain terminator automated sequencing. The sequence that wasdetermined was compared to that of a reference sequence, NL4-3. Thegenotype was examined for sequences that were different from thereference or pre-treatment sequence and correlated to the observed IC₅0for EFV, NVP, and DLV.

As shown in Table 4, genotypic analysis of different clones derived frompatient sample 62 shows that the mutation at codon 369 of the reversetranscriptase of the HIV-1 was present in many clones exhibitingresistance to an NNRTI. In Table 4, “0” means that a particular clonedoes not contain a mutation at the respective codon of the reversetranscriptase of the HIV-1. “1” means that a particular clone contains amutation at the respective codon of the reverse transcriptase of theHIV-1. Five out of six clones containing a mutation at codon 369 showedenhanced resistance. However, one clone containing a mutation at codon369 did not show enhanced resistance. Without intending to be limited toany particular theory, it is believed that this clone comprises one ormore mutations in addition to the mutation at codon 369, which cansuppress NNRTI resistance conferred by the mutation at codon 369.

6.3.2 Mutation at Codon 369 of Reverse Transcriptase of HIV-1 Correlateswith Resistance to an NNRTI

To confirm that a mutation at codon 369 of the reverse transcriptase ofthe HIV-1 significantly correlates with resistance to an NNRTI, asite-directed T369I mutant was generated in an NL4-3 background usingconventional techniques and tested for resistance to NNRTIs using themethods described above.

FIG. 4 demonstrates the T3691 mutation results in enhanced resistance toNVP. Resistance to NVP is shown by the increased 1050 of thesite-directed mutants T369I compared with the control. This result wasalso observed for other NNRTIs. For NVP, the fold change for IC50 was9.74; for DLV, the fold change of for IC₅₀ was 5.00; for EFV, the foldchange for IC50 was 2.99. Taken together, these results indicate thatthe mutation T369I of reverse transcriptase of the HIV-1 significantlycorrelates with reduced susceptibility to an NNRTI.

Further, a site directed double mutant T3691/K103N was generated usingconventional techniques and tested for resistance to an NNRTI using themethods described above. As shown in FIG. 5, T3691/K103N mutantexhibited enhanced resistance to NVP.

In addition, using conventional technique, the T369I mutation wasintroduced into a resistance test vector comprising PR-RT segmentderived from patient sample 62, which comprises mutations A62V, Q102K,K103N, K122E, D123N, C162S, D177E, 1178L, M184V, T200A, Q207E, T215Y,P243S, V245Q, A272P and R277K in reverse transcriptase. Among thesemutations, K103N correlates with resistance to a number of NNRTIs,including NVP. This patient sample was tested for resistance to an NNRTIusing the methods described above. FIG. 6 demonstrates that thisresistance test vector with this genetic background exhibited enhancedresistance to NVP.

Taken together, these resistance assay data show a mutation at codon at369 of reverse transcriptase of the HIV-1 in combination with a mutationat codon 103 significantly correlates with resistance to an NNRTI.

6.4 Example 4 Identifying Additional Mutations Correlated withResistance to an NNRTI

This example provides methods and compositions for identifying mutationsthat correlate with resistance to an NNRTI. Resistance test vectorsderived from patient samples or clones derived from the resistance testvector pools were tested in a resistance assay to determine accuratelyand quantitatively the relative EFV, DLV, or NVP resistance orsusceptibility and replication capacity in the presence or absence ofcertain mutations.

6.4.1 Mutations at Codons 348, 369 and 399 of Reverse Transcriptase ofthe HIV-1 Correlate with Resistance to an NNRTI

To identify additional mutations associated with resistance to an NNRTI,the DLV, EFV, and NVP susceptibility phenotypes for 52 samples,including the 27 samples analyzed in Example 3, were determined usingthe PR-RT and POL amplicons according to Examples 1 and 2. Of thesesamples, two samples exhibited 5-fold different DLV phenotypes, onesample exhibited 5-fold different EFV phenotypes, and five samplesexhibited 5-fold different NVP phenotypes. See FIG. 7. Genotypicanalysis of the samples with discordant phenotypes revealed mutationsfrom wild-type (NL4-3) at, among others, positions 348, 369, and 399.

To confirm the importance of these positions, the number of samples witha mutation at one of these three positions was plotted against FC in NVPsusceptibility. Samples with mutations in each of these three positions,particularly positions 348 and 369, clustered above and FC of 2.0,suggesting that mutations at these three positions might correlate withresistance. See FIG. 8.

6.4.2 Mutations at Codons 348, 369, and 399 of Reverse Transcriptase ofHIV-1 Affect Resistance to NNRTIs

To assess the effects of mutations at positions 348, 369, and 399, aseries of site-directed mutants were constructed in an wild-type (NL4-3)background. The NNRTI susceptibility phenotypes of these mutants werethen determined using the POL segment assay according to Example 2.

In particular, site-directed mutants were constructed in an NL4-3background comprising N348I, T369I, E399D, or the combination of N3481and T369I. The FC in DLV, EFV, and NVP susceptibility for each mutant isshown in FIG. 9. As shown in FIG. 9, N3481, T369I, and the combinationof N3481 and T369I significantly affected susceptibility to all testedNNRTIs (FC>2), but E399D did not (FC<2).

Next, a series of site-directed mutants were constructed to assess theeffects of combinations of N3481, T369I, and E399D with previouslyrecognized NNRTI resistance mutations. In particular, mutants comprisingN348I, T369I, and E399D were made in combination with G190S, with K103N,and with the combination of K103R and V179D. The effects of thesecombinations on DLV, EFV, and NVP susceptibility were tested using thePOL segment assay according to the method describe in Example 2. Theresults from these experiments are presented as FIG. 10.

As shown in FIG. 10, combining any of N3481, T3691, and E399D withG190S, K103N, or the combination of K103R and V179D increased EFV andNVP resistance beyond that observed for G190S, K103N, or K103R and V179Dalone. In fact, the only combination that did not result insignificantly increased NNRTI resistance is the G190S and E399Dcombination in relation to DLV susceptibility. As shown in the table ofFIG. 10, the G190S and E399D combination mutant exhibited the same FC insusceptibility to DLV relative to NL4-3 as the G190S mutant exhibited.

To further analyze the effects of N348I, T369I, and E399D on NNRTIresistance, an additional series of site directed mutants wasconstructed in non-wild-type backgrounds. In particular, site-directedmutants comprising the N348I, T369I, or E399D mutation were constructedin POL-containing test vectors obtained from three different patientsamples. The genotypes of the patient samples were are shown in Table 6,below. Results from these experiments are presented as FIG. 11.

As shown in FIG. 11, addition of N348I and T369I mutations increasedresistance to EFV, NVP, and DLV for all tested patient genotypes. TheE399D mutation increased EFV, NVP, and DLV resistance in a patient 50background, but not a patient 58 or patient 62 background.

6.5 Example 5 Identifying Additional Mutations Correlated withResistance to AZT

This example describes the results of experiments to assess the effectsof the novel NNRTI resistance mutations identified in Example 4, above.

First, the AZT susceptibility phenotypes for 52 samples were determinedusing the PR-RT and POL amplicons according to Examples 1 and 2. Ofthese samples, seven samples exhibited significantly different AZTphenotypes in the two different assays. See FIG. 12.

To assess the specific effects of mutations at positions 348, 369, and399, a series of site-directed mutants were constructed in an wild-type(NL4-3) background. The AZT susceptibility phenotype of these mutantswere then determined with a POL segment assay according to Example 2.

In particular, site-directed mutants were constructed in an NL4-3background comprising N348I, T3691, E399D, or the combination of N348Iand T3691. The FC in AZT susceptibility for each mutant is shown as FIG.13. As shown in FIG. 13, N348I, T3691, and the combination of N3481 andT3691 reduced susceptibility to AZT (FC>2), but E399D did not (FC<2).

To further analyze the effects of N348I, T369I, and E399D on AZTresistance, an additional series of site directed mutants wasconstructed in non-wild-type backgrounds. In particular, site-directedmutants comprising the N3481, T369I, or E399D mutation were constructedin POL-containing test vectors obtained from two different patientsamples. The genotypes of the patient samples are shown in Table 6,below. Results from these experiments are presented in FIG. 14.

As shown in FIG. 14, the N3481 and T369I mutations increased resistanceto AZT in all tested patient backgrounds. The E399D mutation did notsignificantly increase AZT resistance in either the patient 45 orpatient 50 background.

6.6 Example 6 Assessing the Effects of NNRTI Resistance Mutations onReplication Capacity

This example describes the results of experiments to assess the effectsof the NNRTI resistance mutations analyzed in Example 4, above, on thereplication capacity of HIV-1.

First, the replication capacities of certain of the site-directedmutants were determined by performing the phenotypic assay with the POLsegment of Example 2 in the absence of any anti-HIV drug. By comparingthe relative luciferase activity observed for the site directed mutantsrelative to a wild-type reference (NL4-3), the replication capacity ofthe mutants relative to wild-type was determined. The relativereplication capacities for N3481, T3691, E399D, and the combination ofN348I and T369I were determined. Further, the relative replicationcapacities for N348I, T3691, and E399D were determined in the presenceand absence of the G190S mutation and the K103N mutation.

Results of these experiments are presented in FIG. 15. As shown in FIG.15, only T3691 significantly affected replication capacity in theabsence of either G190S or K103N. The effect of the T369I mutation onreplication Capacity was substantially increased by combining it withthe N3481 mutation. Further, in the presence of either G190S or K103N,each of the three mutations N3481, T369I, and E399D further reducedreplication capacity.

In addition, the effects of the N348I, T369I, and E399D mutations onreplication capacity were assessed in the patient backgrounds describedabove using the POL segment assay of Example 2. In particular, thereplication capacities of the site-directed mutants comprising N348I,T369I, and E399D in the genetic background of patient 45, 50, 58, or 62and compared to the patient samples without the mutations. Results arerelative to wild-type and are presented in FIG. 16.

As shown in FIG. 16, the effects of the N348I, T369I, and E399Dmutations on replication capacity were largely dependent on the patientgenetic background. In all tested samples, the additional mutationsreduced replication capacity, but the significance and degree of thatreduction varied by patient. Of the N348I, T369I, and E399D mutations,only the T369I mutation consistently reduced replication capacity.

6.7 Example 6 Identifying Mutations Correlated with AlteredSusceptibility to Integrase Inhibitors

This example provides methods and compositions for identifying mutationsthat correlate with altered susceptibilities to integrase inhibitors.Resistance test vectors derived from patient samples or clones derivedfrom the resistance test vector pools were tested in a resistance assayto determine accurately and quantitatively the relative resistance orsusceptibility of samples to particular integrase inhibitors and thereplication capacity of samples in the presence or absence of certainmutations.

6.7.1 Identification of Mutations Putatively Correlated with AlteredSusceptibility to Integrase Inhibitors

To identify mutations associated with altered susceptibility tointegrase inhibitors, including the integrase strand transfer inhibitor(“INSTI”) L-870,810, the resistance or susceptibility phenotypes for 128patient samples were determined using the RHIN segment as described inExample 2, above. The FC in susceptibility relative to wild-type (NL4-3)was then plotted as a histogram showing the number of samples havingparticular fold-changes in susceptibility, as shown in FIG. 17.

To identify sequence variations that significantly affect susceptibilityto L-870,810, all 128 samples were sequenced using conventionaltechniques. Three samples with increased FC relative to wild-type (i.e.,reduced susceptibility) contained the T97A mutation, while the ninesamples with greatest decrease in FC relative to wild-type (i.e.,increased susceptibility; FC<0.5) contained the K156N mutation.Accordingly, mutations T97A and K156N were further investigated toassess their effects on resistance to integrase inhibitors.

6.7.2 Mutations at Codons 97 and 156 of HIV-1 Integrase AffectResistance to Integrase Inhibitors

To assess the effects of mutations at positions 97 and 156 of integrase,two site-directed mutants (T97A and K156N) were constructed in twodifferent wild-type (NL4-3 and IIIB) genetic backgrounds. The INSTIsusceptibility phenotypes of these mutants to diketo acid 1, diketo acid2, and L-870,810 were then determined using the RHIN segment assayaccording to Example 2.

In particular, site-directed mutants were constructed in an NL4-3 orIIIB background comprising either T97A or K156N. The FC insusceptibility to diketo acid 1, diketo acid 2, and L-870,810 for eachmutant is shown as FIG. 18. As shown in FIG. 18, neither T97A nor K156Nsignificantly affected susceptibility to diketo acid 1 or diketo acid 2in either NL4-3 or IIIB backgrounds. However, T97A resulted in reducedsusceptibility to L-870,810 in both NL4-3 and IIIB backgrounds, whileK156N resulted in increased susceptibility to L-870,810 in bothbackgrounds. Thus, these experiments confirm that T97A is associatedwith resistance to L-870,810 and K156N is associated with increasedsusceptibility to L-870,810.

In addition, the replication capacities for the T97A and K156N mutantsin the NL4-3 background were determined by comparing the luciferaseactivity of the RHIN segments comprising these mutations relative to aNL4-3 RHIN segment in the assay of Example 2 in the absence of an INSTI.As shown in FIG. 19, T97A resulted in significantly reduced replicationcapacity, while K156N did not significantly affect replication capacity.

Next, a series of site-directed mutants were constructed to assess theeffects of combinations of T97A and K156N with previously recognizedINSTI resistance mutations. In particular, mutants comprising T97A,K156N, and the combination of T97A and K156N were made in combinationwith three constellations of previously recognized INSI mutations (V721,F121Y, and T125K; N155S; and T66I and M1541). Each of these combinationswas made in a IIIB background. The effects of these combinations ondiketo acid 1, diketo acid 2, and L-870,810 susceptibility were testedwith the RHIN segment assay according to the method described in Example2. The results from these experiments are presented in FIG. 20.

As shown in FIG. 20, adding T97A, K156N, or the combination of T97A andK156N to the combination V721, F121Y, and T125K, to N155S, or to thecombination T66I and M1541 increased resistance to diketo acid 1 anddiketo acid 2 beyond that observed for the previously recognized INSTIresistance mutations alone. Thus, both T97A and K156N result indecreased susceptibility to diketo acid 1 and diketo acid 2 beyond thatattributable to the previously recognized constellations.

As also shown in FIG. 20, the T97A mutation also resulted in increasedresistance to L-870,810 either alone or in combination with V72I, F121Y,and T125K, with N155S, or with T66I and M1541. K156N resulted in adecrease in L-870,810 FC between about 1.8 and 2.6 fold for thecombinations tested. This effect of K156N was often observed in thepresence of the T97A mutation. Thus, these results demonstrate that T97Aresults in decreased susceptibility to L-870,810, while K156N usuallyincreases susceptibility to L870,810.

All references cited herein are incorporated by reference in theirentireties.

The examples provided herein, both actual and prophetic are merelyembodiments of the present invention and are not intended to limit theinvention in any way.

TABLE 3 Drug Resistance Results of 27 Patient Samples

VP

LV po

/ po

/ Sample PR-RT PR-RT ID po

PR-RT RH

N ratio po

PR-RT RH

N ratio Sample 400.0

8.4 2.1 4.5 28.0 16.4 1.1 1.7 31 Sample 25.1 23.3 1.0 1.1 5.7 8.7 0.70.7 33 Sample 0.3 0.3 0.7 1.3 0.4 0.5 0.7 1.0 34 Sample 28.3 25.2 1.21.1 5.4 6.5 0.8 0.8 35 Sample 2.4 13.0 1.0 0.2 1.9 6.7 0.9 0.2 36 Sample81.0 114.0 0.5 0.7

6.0 128.0 0.5 0.5 38 Sample 2.8 0.3 2.6 9.0 1.2 0.2 1.2 5.1 39 Sample7.1 34.0 0.6 0.2 0.2 0.4 0.5 0.5 42 Sample 0.3 0.3 1.3 1.1 0.1 0.1 1.00.7 45 Sample 400.0 400.0 1.0 1.0 5.4 6.2 0.6 0.

47 Sample 72.7 153.9 0.6 0.5 65.4 250.0 0.4 0.3 50 Sample 0.7 0.4 0.61.6 0.3 0.3 0.4 0.9 51 Sample 1.8 2.0 0.9 0.9 2.7 3.1 0.9 0.8 52 Sample400.0 400.0 1.7 1.0 250.0 149.6 1.5 1.7 58 Sample 400.0 64.0 7.6 6.3

.0 16.0 3.4 8.8 62 Sample 0.3 1.0 0.4 0.3 0.3 1.6 0.3 0.2 64 Sample 1.61.0 0.9 1.6 1.8 1.4 0.7 1.2 69 Sample 1.1 0.7 1.6 1.6 1.2 0.9 1.7 1.4 72Sample 17.0 46.0 0.72 0.4 22.0 65.0 0.58 0.3 80 Sample 114.0 92.0 1.41.2 9.5 12.0 0.9 0.8 81 Sample 0.9 0.8 0.7 1.2 0.5 0.9 0.4 0.6

4 Sample 400.0 131.7 2.1 3.0 69.0 25.2 1.4 2.7 85 Sample 1.4 0.8 1.9 1.70.7 0.7 1.3 0.9

7 Sample 400.0 37.0 2.7 10.8 250.0 250.0 1.5 1.0

9 Sample 400.0 400.0 1.2 1.

4.8 4.1 0.6 1.1 90 Sample 1.1 1.1 0.5 1.0 0.8 1.1 0.3 0.8 92 Sample 1.41.6 1.1 0.9 0.2 0.3 0.7 0.6 93 E

po

/ Sample PR-RT

RTI ID po

PR-RT RH

N ratio Mutations T369 Comments Sample 56.0 22.8 1.1 2.5 K103

31 Sample 5.9 7.1 0.7 0.

K103S 33 Sample 0.6 0.4 1.0 1.4 34 Sample 9.5 9.3 0.7 1.0 K103H 35Sample 2.2 6.7 1.0 0.3 K103

Percentage of 36 K103N in mixture in greater PRRT then po

Sample 27.0 42.0 0.5 0.6 K103

38 Sample 1.0 0.3 0.9 4.0 T369A 39 Sample 1.1 3.1 0.4 0.3 G190G

A 42 Sample 0.2 0.2 1.0 1.

45 Sample 30.2 123.9 0.5 0.2 K103K

, V106 is WT 47 V1

6V

A, in PRPT Y181Y

C, G190G/A Sample 24.1 63.2 0.5 0.4 K103

50 Sample 0.5 0.5 0.4 1.1 51 Sample 1.1 1.3 1.0 0.8 52 Sample 153.9 26.40.9 5.8 K103

, G190 WT 58 Y181Y

C, in PRPT G190G/A Sample 202.0 20.0 1.8 10.1 K103

T369

P225 is WT 62 P225P

H is po

Sample 0.3 1.1 0.3 0.3 T369

64 Sample 1.2 0.8 0.8 1.6 69 Sample 0.8 0.5 1.0 1.5 72 Sample 6.3 11.00.52 0.6 K103K

N, 80 Y181Y

C Sample 136.0 9

.0 1.2 1.4 K103

, 81 P225H Sample 0.7 0.7 0.5 1.0

4 Sample 77.0 22.0 1.2 3.5 85 Sample 1.0 0.9 1.3 1.1

7 Sample 700.0 700.0 1.2 1.0 L10

,

9 K103

Sample 66.0 50.5 0.7 1.3 K103K

N, X103 is WT 90 Y181C, G190A in PRPT Sample 0.8 1.0 0.3 0.8 92 Sample0.7 0.8 0.9 0.8 93

indicates data missing or illegible when filed

TABLE 4 Clonal Analysis of Patient Sample 62 Clone ID NVP FC H315 Y342N348 Y354 A355 T369 A371 T377 I393 T403 N418 R448 D511 K550 c28 0.54 0 00 0 0 0 0 1 1 1 0 1 0 0 c03a 1.64 0 0 0 0 0 0 0 1 0 1 0 0 0 0 c16 2.19 00 0 0 0 0 0 1 0 1 0 0 0.5 0 c15 2.37 0 0 1 1 0 0 0 0 0 1 1 0 0 0 c182.46 0 0 0 0 0 0 1 1 0 1 0 0 0 0 c20 3.97 0 0 0 0 0 0 0 1 0 1 0 0 0 0c24 4.77 0 0 0 0 0 0 0 1 0 1 0 0 0 0 c21 5.16 0 0 0 0 0 0 0 1 0 1 0 0 01 c03b 5.51 0 0 0 0 1 1 0 1 0 1 0 0 0 0 c14 6.48 0 1 0 0 0 0 0 1 0 1 0 10 0 Poo

7.61 0 0 0.5 0 0 0.5 0 1 0 1 0 0 0 0 c12 9.18 0 0 0 0 0 1 0 1 0 1 0 0 00 c23 10.11 0 0 0 0 0 1 0 1 0 1 0 0 0 0 c26 12.00 0 0 0 0 0 1 0 1 0 1 00 0 0 c08 12.98 1 0 0 0 0 1 0 1 0 0 0 0 0 0 c10 28.07 0 0 1 0 0 0 0 1 01 0 0 0 0

indicates data missing or illegible when filed

TABLE 5 Resistance Results with Different Amplicons of Sample 62 (FoldChange in IC₅₀) Amplicon DLV EFV NVP RHIN 3.36 1.83 7.61 pol 140.00202.00 400.00 PRRT 16.00 20.00 64.00 PRRT-RHIN 140.00 202.00 400.00PRRT-RH 250.00 276.90 400.00

TABLE 6 Reverse Transcriptase Genotypes of Patients 45, 50 58, and 62Patient 45 Q23Q/., V35V/I, M41L, K43E, E44A, D67N, T69T/A, V75V/1VI,Q102K, V118I, D123E, C162S, D177E, M184M/V, G196E, Q207K, 11208Y, L210W,R211D/E, T215Y, S251I, A272P, R277K, V293I, K3111C/R, Q334N, K358R,G359G/S, A360T, A371V, K390R, K395R, E399D, A400T, I435V, K454R, P468S,T470D, H483Y, L491S, T497T/S, S519N, K527Q, L533L/M, A554S Patient 50V35T, Q102K, K103N, K122E, C162S, R211K, V245E, I257L, K331K/R, T377K,V381V/I, T386I, K390R, T4031/M, 1435I/A/T/V, E449E/D, V467I, K527N,A554S, L560L/F Patient 58 V35T, M41L, K43E/Q, K49R, A98A/G, Q102K,K103N, V118I, I1351/T, C162S, Y181Y/C, G190G/A, L210W, R211K, T215Y,L228L/H, T240T/K, R277K, T286A, V2931, E297K, V317V/A, 1329L, R356K,IC3581C/R, A360V, T362T/P, E399D, A400T, 1435V, A446S, L452S/T, D460N,P468S Patient 62 W24W/C, A62V, E89E/G, Q102K, K103N, K122E, D123N,1135I/M, C162S, D177E, 1178L, M184V, T200A, Q207E, T215Y, P243P/S,V245Q, A272P, R277K, R284R1G, S322A, N348N/I, K358R, H361H/P, T369T/I,T377M, S379C, A400T, T4031, E404D/N, I435V, L452Q, V4671, P468S, L469I,T470A, K476R, H483Y, S519N, K527N, K530R, A554N

1. A method for determining whether a human immunodeficiency virus 1(HIV-1) is resistant to a non-nucleoside reverse transcriptase inhibitor(NNRTI) or ziduvine (AZT), comprising detecting whether a mutation atcodon 348 or 369 is present in a gene encoding reverse transcriptase ofthe HIV-1, wherein the presence of the mutation correlates withresistance to an NNRTI or to AZT, such that if the mutation is present,the HIV-1 is resistant to the NNRTI or to AZT.
 2. The method of claim 1,wherein the mutation at codon 348 encodes isoleucine (I).
 3. The methodof claim 1, wherein the mutation at codon 369 encodes isoleucine (I) oralanine (A).
 4. The method of claim 1, wherein the NNRTI is efavirenz(EFV), nevirapine (NVP), or delavirdine (DLV).
 5. The method of claim 1,wherein the HIV-1 is determined to be resistant to AZT.
 6. The method ofclaim 1, further comprising detecting whether a mutation at codon 103,179, 190, or 225 is present in the gene encoding reverse transcriptase.7. The method of claim Error! Reference source not found., wherein themutation at codon 103 encodes asparagine (N), arginine (R), serine (S),glutamine (Q), or threonine (T).
 8. The method of claim Error! Referencesource not found., wherein the mutation at codon 225 encodes histidine(H).
 9. The method of claim Error! Reference source not found., whereinthe mutation at codon 103 encodes asparagine (N), arginine (R), serine(S), glutamine (Q), or threonine (T).
 10. The method of claim Error!Reference source not found., wherein the mutation at codon 225 encodeshistidine (H).
 11. The method of claim Error! Reference source notfound., wherein the mutation at codon 190 encodes serine (S).
 12. Themethod of claim Error! Reference source not found., wherein the mutationat codon 103 encodes asparagine (N), arginine (R), serine (S), glutamine(Q), or threonine (T).
 13. The method of claim Error! Reference sourcenot found., wherein the mutation at codon 179 encodes aspartic acid (D).14. The method of claim 6, wherein the NNRTI is EFV, NVP, or DLV.
 15. Amethod for determining whether a human immunodeficiency virus 1 (HIV-1)is resistant to a non-nucleoside reverse transcriptase inhibitor(NNRTI), comprising detecting whether a mutation at codon 399 incombination with a mutation at codon 103, 179, or 190 is present in agene encoding reverse transcriptase of the HIV-1, wherein the presenceof the mutations correlates with resistance to an NNRTI, such that ifthe mutation is present, the HIV-1 is resistant to the NNRTI.
 16. Themethod of claim 15, wherein the mutation at codon 399 encodes asparticacid (D).
 17. The method of claim Error! Reference source not found.,wherein the mutation at codon 103 encodes asparagine (N), arginine (R),serine (S), glutamine (Q), or threonine (T).
 18. The method of claimError! Reference source not found., wherein the mutation at codon 179encodes aspartic acid (D).
 19. The method of claim Error! Referencesource not found., wherein the mutation at codon 190 encodes serine (S).20. The method of claim 6, wherein the NNRTI is EFV, NVP, or DLV.
 21. Amethod for determining whether an HIV-1 has reduced replication capacityrelative to a reference HIV-1, comprising detecting, in a nucleic acidencoding reverse transcriptase of the HIV-1, a mutation at codon 369 orin a nucleic acid encoding integrase of the HIV-1, a mutation at codon97, wherein the presence of the mutation correlates with reducedreplication capacity such that if the mutation is present, the HIV-1 hasreduced replication capacity relative to a reference HIV-1.
 22. Themethod of claim Error! Reference source not found., wherein the mutationat codon 369 encodes isoleucine (I).
 23. The method of claim Error!Reference source not found., wherein the mutation at codon 97 encodesalanine (A).
 24. The method of claim 21, wherein the reference HIV-1 isNL4-3.
 25. The method of claim 21, wherein the reference HIV-1 has anidentical genotype to the HIV-1 having its replication capacitydetermined except for codon 369 of reverse transcriptase or codon 97 ofintegrase.
 26. A method for determining whether a human immunodeficiencyvirus 1 (HIV-1) has altered susceptibility to a integrase strandtransfer inhibitor (INSTI), comprising detecting whether a mutation atcodon 97 or codon 156 is present in a gene encoding integrase of theHIV-1, wherein the presence of the mutations correlates with alteredsusceptibility to an INSTI, such that if the mutation is present, theHIV-1 has altered susceptibility to the INSTI.
 27. The method of claim26, wherein the HIV-1 exhibits increased susceptibility to the INSTI.28. The method of claim 26, wherein the HIV-1 exhibits decreasedsusceptibility to the INSTI.
 29. The method of claim Error! Referencesource not found., wherein the mutation at codon 97 encodes alanine (A).30. The method of claim Error! Reference source not found., wherein themutation at codon 156 encodes asparagines (N).
 31. The method of claim26, wherein the INSTI is a napthyridine carboximide.
 32. The method ofclaim 26, wherein the INSTI is L-870,810.
 33. The method of claim 26,further comprising detecting whether a mutation at codon 66, 72, 121,125, 154, or 155 is present in the gene encoding integrase.

HIV-1 exhibits decreased susceptibility to the INSTI.
 35. The method ofclaim 33, wherein the mutation at codon 66 encodes isoleucine (I). 36.The method of claim 33, wherein the mutation at codon 72 encodesisoleucine (I).
 37. The method of claim 33, wherein the mutation atcodon 121 encodes tyrosine (Y).
 38. The method of claim 33, wherein themutation at codon 125 encodes lysine (K).
 39. The method of claim 33,wherein the mutation at codon 154 encodes isoleucine (I).
 40. The methodof claim 33, wherein the mutation at codon 155 encodes serine (S). 41.The method of claim 33, wherein the INSTI is a diketo acid.
 42. Themethod of claim 26, wherein the INSTI is a napthyridine carboximide. 43.The method of claim 26, wherein the INSTI is L-870,810.